Crafting of cartilage

ABSTRACT

A shaped cartilage matrix isolated from a human or animal where the cartilage has been crafted to facilitate disinfection, cleaning, devitalization, recellularization, and/or integration after implantation. Also, a process for repairing a cartilage defect with the cartilage matrix. The matrix is in the form of an osteochondral plug including a cartilage cap ad subchondral bone, wherein one or more gaps, slats, bores, or channels extend through the tidemark at the interface between the cartilage cap and the subchondral bone.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of and claims priority under 35U.S.C. § 120 to U.S. Ser. No. 11/826,518, filed Jul. 16, 2007, theentire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention is directed to producing a shaped cartilage matrixisolated from a human or animal where the cartilage has been crafted tofacilitate disinfection, cleaning, devitalization, recellularization,and/or integration after implantation.

BACKGROUND OF THE INVENTION

Cartilage is a highly hydrated connective tissue with chondrocytesembedded in a dense extracellular matrix made of, for example, collagen,proteoglycan and water. Although the biochemical composition ofcartilage differs according to types, there are mainly three types ofcartilage present in a mammal, which include: articular or hyalinecartilage, fibrocartilage, and elastic cartilage. Hyaline cartilage ispredominantly found on the articulating surfaces of articulating jointsand contains type II collagen and proteoglycans. It is found also inepiphyseal plates, costal cartilage, tracheal cartilage, bronchialcartilage, and nasal cartilage. Fibrocartilage is mainly found inmenisci, the annulus fibrosis of the intervertebral disc, tendinous andligamentous insertions, the symphysis pubis, and insertions of jointcapsules. The composition of fibrocartilage is similar to hyalinecartilage except that fibrocartilage contains fibrils of type I collagenthat add tensile strength to the cartilage. Elastic cartilage is presentin the pinna of the ears, the epiglottis, and the larynx and is similarto hyaline cartilage except that it contains fibers of elastin.

One of the most common cartilage injuries is damage to thefibrocartilage in the knee joint. Meniscal tears are common in youngindividuals due to sports-related injuries, as well as in olderpopulation suffering from degenerative joint diseases. Meniscalallograft transplantation is one of the available treatment options forpatients with meniscal tear. Despite some positive results, issues withtissue rejection, disease transmission and a lack of long-term data havelimited the use of this approach.

Diseased or traumatized intervertebral disc is another commonfibrocartilage injury. The damage on the annulus can cause pain andpossible disc herniation that can compress nerves or the spinal cordresulting in arm or leg pain and dysfunction. Recent advances inmolecular biology, cell biology and material sciences have opened a newemerging field for cartilage repair.

However, the most common cartilage injury is articular cartilage injuryoften as a result of sports related trauma. Due to its avascular nature,articular cartilage has very limited capacity for repair. Approximately500,000 arthroplastic or joint repair procedures are performed each yearin the United States. These procedures include approximately 125,000total hip and 150,000 total knee arthroplastic procedures (Chen, et al.,Repair of articular cartilage defects: Part 1, Basic Science ofArticular Cartilage Healing, Amer. J. Orthopedics 1999:31-33). Articularcartilage is a complex tissue involving biomechanical function andassociated physical stimuli inside the articular cartilage. Articularcartilage is an inhomogeneous material (tissue) and surface loading isconverted to mechanical and electrochemical signals by the extracellularmatrix through hydraulic and osmotic pressures, fluid and solute/ionflows, matrix deformations and electrical fields (Mow, Van C. and C.C-B. Wang, Some bioengineering considerations for tissue engineering ofarticular cartilage. Clinical and Orthopedics and Related Research.1999, Number 367s, S204-S223).

Unfortunately, chondral defects may not heal, especially when the defectdoes not penetrate the subchondral bone. A wide variety of surgicalprocedures are in current use or have been proposed for use to repairchondral defects attempt to prompt the resident cellular population tobecome more metabolically active thereby promoting new matrix synthesis,however, for the most part, these surgical procedures do little morethan provide temporary relief of pain.

SUMMARY OF THE INVENTION

One aspect of the invention is to produce a devitalized and shapedcartilage graft suitable for recellularizing in vitro, in vivo, or insitu. The devitalized cartilage graft, particularly cartilage graft, maybe derived from articular cartilage of human or other animal(s) in someaspects. The subchondral bone, i.e., the cancellous bone portion of thegraft, if present, may be cleaned and disinfected to remove bone marrowelements, and the cartilage portion of the graft may be made a cellular.Furthermore, the subchondral bone portion may be crafted into varioussizes and shapes and modified to incorporate gaps, a bore, channels, orslots to render cleaning, disinfection, devitalization, andrecellularization. The cartilage part of the graft may be treated toimprove recellularization by chemical or physical modification. Thecartilage may further be recellularized from devitalized cartilagematrix. Moreover, the cartilage graft may be implanted into a recipientand sealed with recipient tissue.

The present invention relates to a cartilage matrix isolated from ahuman or animal where the cartilage matrix may be crafted, cleaned, anddisinfected. A further aspect of the invention relates to a shapedcartilage matrix isolated from a human or animal, wherein the cartilagemay be crafted, cleaned, disinfected, and devitalized. An even furtheraspect of the invention relates to a shaped cartilage matrix isolatedfrom a human or animal, wherein the cartilage may be crafted, cleaned,disinfected, devitalized, and recellularized. A further aspect of theinvention relates to the aforementioned shaped cartilage matrix whichfacilitates the integration after implantation.

The invention further relates to a shaped cartilage matrix isolated froma human or animal where the cartilage has been crafted to facilitatedisinfection, cleaning, devitalization, recellularization, and/orintegration after implantation. The cartilage matrix may be isolatedfrom whole condyles, whole plateaus, hemicondyles, hemiplateaus, femoralheads, phalanges, talus, tibia, fibula, rib, intervertebral discs,menisci, nose, or an ear. The cartilage matrix may be in the form ofwhole condyles, whole plateaus, hemicondyles, hemiplateaus, femoralheads, phalanges, talus, tibia, fibula, rib, intervertebral discs,menisci, nose, ear, osteochondral plugs, cartilage discs, cartilageslices, cartilage curls, or cartilage flakes.

A further aspect of the invention relates to a process for repairing acartilage defect and implantation a cartilage graft into a human oranimal by optionally crafting the cartilage matrix into individualgrafts, disinfecting and cleaning the cartilage graft, applying apretreatment solution to the cartilage graft, removing cellular debrisusing an extracting solution to produce a devitalized cartilage graft,implanting the cartilage graft into the cartilage defect with or withoutan insertion device, and sealing the implanted cartilage graft withrecipient tissue. The devitalized cartilage graft is optionallyrecellularized in vitro, in vivo, or in situ with viable cells to renderthe tissue vital before or after the implantation. The devitalizedcartilage graft is also optionally stored between the removing cellulardebris and the recellularizing steps.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a view of a knee joint that is processed to havearticular cartilage grafts of (a) whole condyle, whole plateau,hemicondyles, hemiplateaus, or (b) osteochondral plugs.

FIG. 2 illustrates an enlarged view of the cylindrical shapedosteochondral plugs with subchondral bone attached. The subchondral boneportion is crafted to have gaps or channels or slots. The last row ofthe figure shows the bottom view of the osteochondral plug.

FIG. 3 illustrates an enlarged view of the dumbbell shaped osteochondralplugs with subchondral bone attached. The subchondral bone portion iscrafted to have gaps or channels or slots. The last row of the figureshows the bottom view of the osteochondral plug.

FIG. 4 illustrates an enlarged view of the step cylindrical shapedosteochondral plugs with subchondral bone attached. The subchondral boneportion is crafted to have gaps or channels or slots. The last row ofthe figure shows the bottom view of the osteochondral plug.

FIG. 5 illustrates an enlarged view of the osteochondral plugs or discsthat are cut into two halves or four quarters along the diameter of theplug.

FIG. 6 illustrates an enlarged view of the osteochondral plugs where thecircumferential surface of the cartilage caps is crafted to increase thesurface area. The cartilage discs of full depth cartilage are obtainedby cutting the crafted cartilage caps off the osteochondral plugs.

FIG. 7 illustrates a view of an osteochondral plug holder for craftingfrom the subchondral bone portion from the bottom to obtain more thanone gaps that form angles between 0-180 degrees; or to obtain a hollowcylinder; or obtain multiple channels along the entire length of thesubchondral bone portion up to the cartilage and subchondral boneinterface.

FIG. 8 illustrates a view of an osteochondral plug holder for craftingthe cylindrical surface of the subchondral bone portion at the cartilageand subchondral bone interface to obtain more than one channels (13)that form 0-90 degree angles.

FIG. 9 illustrates a view of an osteochondral plug holder for craftingthe cylindrical surface of the subchondral bone portion at the cartilageand subchondral bone interface to form multiple parallel through holesor channels or a slot.

FIG. 10 illustrates an assembly of a cutting device, where a star-shapedcutting blade (65) is fit into an adaptor (66) and used to cut astar-shaped cartilage cap on the osteochondral plug. Then a pushingdevice (67) is used to push out the osteochondral plug from theadaptor/cutting blade assembly.

FIG. 11 illustrates a star-shaped cutting blade to cut a star-shapedcartilage cap on an osteochondral plug.

FIG. 12 illustrates an adaptor for the cutting blade.

FIG. 13a illustrates a view of one embodiment of a cleaning/processingchamber (75) that can be fit into a centrifugation device. Cartilagegrafts are fit into an insert (80) and the processing fluid is forcedthrough the cartilage graft during centrifugation.

FIG. 13b illustrates a top and a side view of an insert (80), that theosteochondral plugs are fit into so that the superficial area of thecartilage surface is perpendicular to the fluid flow direction.

FIG. 14 illustrates a view of one embodiment of a cleaning/processingchamber (75). Cartilage grafts are fit into an insert (80) andprocessing fluid is forced through the cartilage graft using vacuumpressure.

FIG. 15 illustrates a pressurized flow through devitalization systemwhere fluids are recirculated between a cleaning/processing chamber (96)with insert (101) and a reservoir. The superficial surface of thecartilage graft is perpendicular to the fluid flow direction.

FIG. 16a illustrates a pressurized flow through devitalization systemwhere fluids are recirculated between a cleaning/processing chamber (96)with an insert (274) and a reservoir.

FIG. 16b illustrates a top and a side view of an insert (274) that thecartilage grafts are fitted in so that the superficial surface of thecartilage grafts are parallel to the fluid flow direction.

FIG. 17a illustrates a cyclic hydrodynamic pressurized devitalizationsystem where the fluid is cyclically pressurized within thecleaning/processing chamber (96).

FIG. 17b illustrates a top and a side view of an insert (118) where awell (124) is interchangeable within the step cylindrical hole (119) toaccommodate a different thickness of the cartilage disc or a stack ofcartilage slices to create a contoured cartilage graft.

FIG. 18a illustrates an embodiment of a packaging device where cartilagegrafts are immersed in a storage solution.

FIG. 18b illustrates an embodiment of packaging device where excessstorage solution is removed and the wet cartilage grafts are packagedwith or without vacuum and stored.

FIG. 19 illustrates an enlarged view of a procedure of recellularizationof the cartilage discs or slides in situ and implantation of therecellularized cartilage graft with a filler to form a composite graftto repair an osteochondral defect.

FIG. 20 illustrates an enlarged view of a procedure of creating acontoured cartilage graft. Devitalized and/or recellularized cartilageslides with varying diameters are stacked to match the curvature of therecipient tissue.

FIG. 21 illustrates the components of a bioreactor. The components areassembled to become the bottom chamber of a bioreactor for in vitrorecellularization and cultivation of a devitalized cartilage graft.

FIG. 22 illustrates the components of a bioreactor. The components areassembled to become the top portion of a chamber of a bioreactor for invitro recellularization and cultivation of a devitalized cartilagegraft.

FIG. 23 illustrates a bioreactor assembly where sterile filtered air iscompressed cyclically towards two gas permeable flexible membranes (193and 172), which induce pressure on a cartilage graft sandwiched betweentwo porous platens within a confining ring in a bioreactor filled withculture media.

FIG. 24 illustrates a bioreactor assembly where fluid within thebioreactor is pressurized cyclically to induce pressure on a cartilagegraft sandwiched between two porous platens within a confining ring in abioreactor filled with culture media.

FIG. 25 illustrates a bioreactor assembly wherein a cartilage graftsandwiched between two porous platens within a confining ring iscompressed with a compression shaft connected to a damping spring.

FIG. 26 illustrates a bioreactor assembly that is positioned vertically.The cartilage cap and the bone portion of a devitalized osteochondralplug are recellularized separately with the same or different cell typesin a bioreactor.

FIG. 27 illustrates a bioreactor assembly that is positionedhorizontally. The cartilage cap and the bone portion of a devitalizedosteochondral plug are recellularized at the same time with the same ordifferent cell types in a bioreactor.

FIG. 28 illustrates a bioreactor assembly wherein a cartilage cap of anosteochondral plug is sandwiched between two porous platens within aconfining ring and is compressed with a compression shaft with orwithout a damping spring connected.

FIG. 29 illustrates a bioreactor assembly wherein the cartilage caps oftwo osteochondral plugs are placed opposite each other within aconfining ring and are compressed with a compression shaft.

FIG. 30 illustrates a bioreactor assembly wherein the cartilage caps oftwo osteochondral plugs are placed opposite each other to createcongruent surfaces within a confining ring and is compressed with acompression shaft. Alternatively, a mold with a target curvature arecompressed against a cartilage cap of an osteochondral plug.

FIG. 31a illustrates a view of a procedure during open knee surgerywherein a cutting device is pushed into the cartilage portion ofrecipient defect site after a straight bore has been created usingconventional surgical tools.

FIG. 31b illustrates a view of a procedure during open knee surgerywherein the adaptor is released from the cutting device and astar-shaped cutter remains in the recipient defect site. The star-shapedcutter is used as a boundary for removing the damaged cartilage from therecipient cartilage to create a star-shaped bore in the cartilageportion of the recipient defect site.

FIG. 32 illustrates a star-shaped cutting blade to create a star-shapedbore in the cartilage portion of the recipient defect site.

FIG. 33 illustrates a view of one embodiment of an insertion device forsurgical insertion of osteochondral plugs, cartilage discs, or a stackof cartilage slices into a bore created at a defect site.

FIG. 34 illustrates a view of a procedure during open knee surgerywherein the shaped cartilage bore and the circumferential area of acartilage cap on an osteochondral plug or a cartilage disc is treatedwith a photoactive dye before insertion of the cartilage graft into theshaped bore. An energy source is applied to seal the cartilageinterface.

FIG. 35a illustrates a view of a procedure during arthroscopic minimallyinvasive surgery wherein a cutting device is pushed into the cartilageportion of recipient defect site after a straight bore has been createdusing conventional surgical tools.

FIG. 35b illustrates a view of a procedure during arthroscopic minimallyinvasive surgery wherein the adaptor is released from the cutting deviceand a star-shaped cutting blade remains in the recipient defect site.The star-shaped cutting blade is used as a boundary for removing thedamaged cartilage within the boundary from the recipient cartilage tocreate a star-shaped bore in the cartilage portion of the recipientdefect site.

FIG. 36 illustrates a procedure during arthroscopic minimally invasivesurgery wherein the shaped cartilage bore and the circumferential areaof a cartilage cap on an osteochondral plug or a cartilage disc istreated with a photoactive dye before insertion of the cartilage graftinto the shaped bore.

FIG. 37 illustrates a procedure during arthroscopic minimally invasivesurgery wherein an energy source is applied to seal the cartilageinterface.

FIG. 38 illustrates the percentage of DNA and proteoglycan reduction incartilage discs after devitalization with 0.5% CHAPS in combination withor without pretreatment with chondroitinase ABC.

FIG. 39 illustrates the H&E and Safranin O staining of cartilage discsafter devitalization with 0.5% CHAPS in combination with or withoutpretreatment of chondroitinase ABC.

FIG. 40 illustrates a procedure for a coating growth factor on thecartilage portion of an osteochondral plug.

FIG. 41 illustrates a procedure for a coating growth factor on theentire osteochondral plug.

DESCRIPTION OF THE INVENTION

The terms “autologous” (autograft) and “allogenous” (allograft) are usedto describe tissues derived from the individual to receive the tissueand tissues derived from an individual other than the individual fromthe same species to receive the tissue, respectively.

The phrase “cleaning solution” is used to describe a solution to cleanallografts, xenografts, and autografts. The phrase cleaning solution isfurther meant to describe any cleaning solution which may be used toclean and/or disinfect these tissues.

The phrase “decontaminating agent” is used to describe any substancewhich can be used to decontaminate bone and/or cartilage. Suchsubstances include, but are not limited to, one or more agents whichremove or inactivate/destroy any infectious material. Non-exclusiveexamples of decontaminating agents include antibacterial agents,antiviral agents, and antimycotic agents. Moreover, the phrasedecontaminating agents is also meant to include, but is not limited tosubstances which may clean bone and/or cartilage by inactivating one ormore of bacteria, viruses, and/or fungi such as hydrogen peroxide,detergents, and alcohols. Further examples of decontaminating agentsinclude acids such as hydrochloric acid and bases such as hydrogenperoxide.

The term “devitalized” involves the decellularization, or making tissueacellular, such that minimal cellular remnants remain.

The phrase “recellularizable cells” means cells capable ofrecellularizing a matrix. Examples of such cells include, but are notlimited to autologous or allograft chondrocytes isolated from articularcartilage, fibrocartilage, or elastic cartilage; bone marrow aspirate;or stromal cells from bone marrow, synovium, periostieum, perichondrium,muscle, dermis, umbilical cord blood, adipose tissue, or Warton's jelly;or pericytes.

Individual grafts may be crafted out of bone plugs with associatedcartilage. Load-bearing osteoimplants may comprise a shaped, compressedcomposition of bone particles for repairing bone. Moreover, cartilagemay be combined with associated cancellous bone in the form of plugs forthe repair of cartilage defects. Further, cartilage gel or paste may beinserted in the space between the plug and the wall of the hole createdin the cartilage to be repaired. A paste or gel sterile implant materialmay be made of milled lyophilized allograft cartilage pieces in abioabsorbable carrier for a cartilage defect. Even further, plugs may becreated with a base membrane, a control plug, and a top membrane whichoverlies the surface of the cartilage covering the cartilage defect andan allograft plug with a cartilage cap which is surface contour matchedto the surface of the cartilage defect area to be replaced,respectively.

Demineralized bone matrix (DBM) has substantial osteoconductive activityand may stimulate new bone growth. The intrinsic growth factors in theDBM can recruit recipient progenitor cells and affect thedifferentiation of the recruited cells into not only osteogenic but alsochondrogenic lineage. When DBM was used as a subchondral matrix in acartilage repair model, subchondral bone growth may be stimulated. Inaddition, the perichondrium (if used) can be fixed on the DBM to providecellular source. Because DBM is less deformable and not quicklyresorbed, it is believed that it could provide mechanical support to theneocartilage formed on the top, at the beginning of the repair, togetherwith the promotion of bone healing in the bottom of the osteochondraldefect. Alternatively, crushed cartilage and cancellous bone fromnon-load bearing region may be used for cartilage repair.

One aspect of the present invention is directed to the repair ofcartilage using cartilage grafts crafted, cleaned, disinfected,devitalized, and optionally recellularized. The devitalized cartilagegrafts may be made sterile and preserved using various methodologies.Large devitalized cartilage grafts such as a hemicondyle may be fittedinto the surgical site appropriate to the articulation needed tomaximize interaction with the opposing cartilage on the bone inapposition to the graft being inserted. Small devitalized osteochondralplugs may be compression fitted into bores drilled into, and coveringthe cartilage defect such that the cancellous bone part of the graftfits tightly into the bore created using conventional surgical tools andthe cartilage part of the graft may be slightly compressed around itsperimeter as it is press fitted into the bore. The cartilage part of thegraft should be at the same height as the surrounding cartilage of therecipient. The cartilage may be sectioned into slices parallel to thearticular surface with various thicknesses. Different sizes and shapesof cartilage can be used to build various contour of the cartilagesurface or have cells seeded to regenerate viable cell population incartilage grafts. The cartilage grafts can also be skived or shaved intocurls or flakes with irregular shapes. The cartilage curls and/orcartilage flakes can be mixed with or without a matrix and/or a carrierto become a filler to fill the cartilage defects. In addition, thecartilage curl and/or cartilage flake filler can be applied incombination with a cartilage slice or a cartilage disc or anosteochondral plug to repair a cartilage defect.

The present invention is directed to an cartilage component (part) of agraft which may be made acellular (devitalized) using one or moredetergents, enzymes to modify the molecular aspects of the cartilage,and a recombinant endonuclease, for example BENZONASE® (Merk, Inc.). Thedevitalized graft may be processed to remove residuals of devitalizationreagents sufficient to render the graft biocompatible, biohospitable,and recellularizable.

The present invention is also directed to a method and process ofclinical use of cartilage components as grafts wherein the surface areasbetween the recipient and the implanted cartilage graft may be maximizedand the interface between the recipient and the implanted cartilagegraft may be molecularly cross-linked to control fluid movement when therepaired tissues may be subjected to loading as would occur duringnormal physiological activities such as, but not restricted to, walking,standing, sitting, running, jogging, or sleeping.

The human femoral condyles, tibial plateaus or femoral heads may beprocured from a suitable donor, transported on wet ice to the processingfacility, processed as whole or bisected into two hemicondyles orhemiplateaus, or cored out to obtain multiple osteochondral plugs asillustrated in FIG. 1. The orientation and anatomical location of thecartilage graft residing on the donor tissue can be recorded using agrid and a coordinate system so that it can be matched to theorientation and anatomical location of the recipient tissue. Theosteochondral plug (5) can be crafted so that the diameter of thesubchondral bone portion (7) is the same as that of the cartilage cap(6) to form a straight cylinder as illustrated in FIG. 2. Alternatively,the diameter of the subchondral bone portion (7) right underneath of thecartilage cap can be made to be slightly smaller than the cartilage cap(7) to form a dumbbell shape as illustrated in FIG. 3; or the diameterof the cartilage cap and the portion of the subchondral bone directlycontacted with the cartilage cap can be in the same diameter as thebottom part of the subchondral bone portion, and the part of thesubchondral bone portion between the bottom part and the portiondirectly contacted with the cartilage cap of the subchondral bone can beslightly smaller in diameter than the rest of the osteochondral plug toform a dumbbell shape. Furthermore, the diameter of the subchondral boneportion (7) can be made to be slightly smaller than the cartilage cap(6) to form a step cylindrical shape as illustrated in FIG. 4; or thediameter of the cartilage cap and the portion of the subchondral bonedirectly contacted with the cartilage cap can be slightly larger thanthe rest of the bone portion to form a step cylindrical shape. Inaddition, as illustrated in FIG. 2, the osteochondral bone portion (7)of the osteochondral plug (5) can be crafted into plugs (8 a, 8 b, 10,12, 14, 16 or 18) to expose one or more portions of the cartilage cap(6) at the cartilage/bone interface. In one embodiment, portion of thetidemark at the cartilage and subchondral bone interface can be removedto expose one or more portions of the cartilage cap at thecartilage/bone interface. In another embodiment, one or more portions ofthe cartilage at the cartilage and subchondral bone interface can beremoved along with the tidemark. In yet another embodiment, the portionof the circumferential area of the cartilage cap that is directlycontacting the subchondral bone can be separated from the subchondralbone at the tidemark to allow the cartilage cap to deform laterallyduring compression in vivo.

Many methods can be used to craft osteochondral plugs, the followingexamples are representative examples and are not meant to be limiting inany respect. Osteochondral plugs of the present invention may have alength of between about 1 and 20 mm and 8 and 20 mm and may have adiameter at its widest point of between about 8 and 20 mm As illustratedin FIG. 2, the osteochondral plug (8 a) can be made by cutting thecylindrical bone portion (7) to obtain one or more gaps (9) that formangles between about 0 to about 180 degrees along the entire length ofthe bone portion up to the cartilage and osteochondral bone interface.The gaps may occupy one or more portions of the cartilage cap directlyin contact with the subchondral bone, and may end at the deep, middle,or superficial zone of the cartilage cap along the cartilage depthdirection and do not penetrate the superficial surface of the cartilagecap. The gaps can also be crafted parallel to the center line of theosteochondral plug and parallel to each other (8 b). The width of thegaps can be between about 1/10 and about ½ of the diameter of the boneportion (7). The osteochondral plug (10) can be obtained bydrilling/milling from bottom of the bone portion (7) along the centerline to form a hollow cylinder. The hollow cylinder has a blind endcenter bore (11) that is along the whole length of the subchondral boneportion and ends at the cartilage and subchondral bone interface. Theblind end center bore (11) may also occupy one or more portions of thecartilage cap directly contacted with the subchondral bone and may endat the deep, middle, or superficial zone of the cartilage cap along thecartilage depth direction and may not penetrate the superficial surfaceof the cartilage cap. The diameter of the blind end center bore (11) ofthe hollow cylinder ranges from about ½ to about ⅘ of the diameter ofthe subchondral bone portion of the osteochondral plug. Theosteochondral plug (12) can be obtained by drilling/milling on thecylindrical surface of the bone portion (7) at the cartilage/boneinterface to form one or more channels (13) that form about 0 to about90 degree angles. The channel width may be from about 1/10 to about ½ ofthe diameter of the subchondral bone portion of the osteochondral plug.The channels may occupy one or more portions of the deep and/or middlezone of the cartilage cap along the depth direction and may not occupythe superficial zone of the cartilage cap. The osteochondral plug (14)can be obtained by drilling/milling from bottom of the bone portion (7)to form multiple about 0.5 to about 1 mm diameter channels (15) alongthe whole length of the bone portion up to the cartilage andosteochondral bone interface. The channels may occupy one or moreportions of the cartilage cap directly contacted with the subchondralbone, may end at the deep, middle, or superficial zone of the cartilagecap along the cartilage depth direction, and may not penetrate thesuperficial surface of the cartilage cap. The osteochondral plug (16)can be obtained by drilling/milling through the cylindrical surface ofthe bone portion (7) at the cartilage/bone interface to form multipleparallel about 0.5 to about 1 mm diameter channels (17). The channelshave the length going through the entire diameter of the subchondralbone portion. The channels may occupy one or more portions of the deepand/or middle zone of the cartilage cap along the depth direction andmay not occupy the superficial zone of said cartilage cap. Osteochondralplug (18) can be obtained by drilling/milling through the cylindricalsurface of the bone portion (7) at the cartilage/bone interface to formone or more slots (19). The slots may have the depth going through theentire diameter of the subchondral bone portion, the height being about0.35 to about 3 mm, and the width being about 1/10 to about ⅘ of thediameter of the subchondral bone of the osteochondral plug. The slotsmay occupy one or more portions of the deep and/or middle zone of thecartilage cap along the depth direction and may not occupy thesuperficial zone of the cartilage cap.

Similarly, as illustrated in FIG. 3, the osteochondral bone portion (21)of the dumbbell shape osteochondral plug (20) can be crafted into plugs(22 a, 22 b, 23, 24, 25, 26, or 27) to expose one or more portions ofcartilage cap (6) at the cartilage/bone interface using the samecrafting procedures described above in FIG. 2. In addition, asillustrated in FIG. 4, the osteochondral bone portion (29) of the stepcylindrical shape osteochondral plug (28) can be crafted into plugs (22a, 22 b, 23, 24, 25, 26, or 27) to expose one or more portions ofcartilage cap (6) at the cartilage/bone interface using the samecrafting procedures described above in FIG. 2. If desired, a cartilagedisc (6) without the subchondral bone portion attached can be obtainedby carefully cutting off the bone portion. The cartilage cap (6) of anosteochondral plug can also be sectioned into thin slices (127) withthicknesses ranging from about 50 to about 1000 μm as illustrated inFIG. 4. These cartilage slices can be trimmed to have circular, square,triangular, or star shapes. These slices can also be trimmed to haveascending or descending diameters and may be stacked together to createa contour that matches the contour of the defect site as illustrated inFIG. 20. The osteochondral plugs, cartilage disc, or cartilage slicesdescribed above can be further cut into two halves or four quartersalong the diameter of the grafts as illustrated in FIG. 5.

The cartilage matrix can also be skived, grated or shaved using a bonefiber shaving device as illustrated in U.S. Patent Application Number20040059364 to produce cartilage flakes or cartilage curls. This patentapplication is hereby incorporated by reference in its entirety. Thecartilage tissue, such as a femoral condyle, can be fixed on a fixtureunderneath of a blade mounted in a cutter. The cutter moves horizontallyrelative to the cartilage tissue during a cutting stroke. The size andthickness of the cartilage flakes or curls can be controlled byadjusting the height of the cutter, the cutting angles, and the distanceof each stroke relative to the cartilage tissue. The size of thecartilage flake or curl can be from about 0.001 to about 10 cm³, about0.001 to about 1 cm³, about 0.01 to about 1 cm³, about 0.1 to about 1cm³.

The circumferential area of the cartilage portion of an osteochondralplug or a cartilage disc can be further crafted to maximize thecircumferential surface and contact areas between the recipientcartilage being repaired and the cartilage graft, as illustrated in FIG.6, to facilitate integration of the graft tissue to the recipienttissue. The surface area maximization can be conducted on anon-devitalized cartilage graft, or a devitalized cartilage graft, or adevitalized and recellularized cartilage graft. The star-shapedcartilage disc (37) or the star-shaped cartilage cap on osteochondralplug (36) can be obtained by coring a cartilage cap (6) with a custommade star-shaped cutting device as illustrated in FIG. 10-FIG. 12. Thecoring device may be composed of a star-shaped cutter (65) and anadaptor (66) (FIG. 10). The size and shape of the star-shaped cuttermatches the size and shape of the star-shaped bore created in the defectsit. The star-shaped cutter may be designed so that its inner surfacemay be straight and the bottom portion of its outer surface may beangled to form a beveled sharp cutting edge FIG. 11. The adaptor (66)may be designed to have slots (73) that can fit into theteeth/protrusions of the stars on the star-shaped cutter (FIG. 12). Theadaptor can also have four slits (273) to allow slight expansion of theadaptor when it fits into the star-shaped cutter. During application,the star-shaped cutter with the assist of the adaptor can punch and cutthrough the cartilage tissue from the osteochondral side or thesuperficial surface side of the cartilage graft. Then the cartilagegraft can be removed from the coring device with the assistance of apushing device (67). Optionally, if the cutting may be performed in theoperating room right before the implantation, the star-shaped cartilagegraft can be maintained in the cutter until implantation to preventlateral expansion.

The tapered cylindrical cartilage disc with (38) or without (39)subchondral bone attached can be obtained using a lathe and an angledcutting tool. The diameter of the superficial region of the taperedcylindrical cartilage cap or disc (39) can be larger than the diameterof the deep region that may be connected to the subchondral bone. Thestraight cylindrical cap (6) or a tapered cylindrical cap (39) can befurther crafted to maximize circumferential surface area by embossingwith a die that has a straight or non-straight line pattern (40 and 41)or cross-line pattern (42 and 43). The straight cylindrical cap (6) or atapered cylindrical cap (39) can also be further crafted to maximize thecircumferential surface area by spraying or blasting microparticles ontothe circumferential surface (44). The microparticles may be selectedfrom a group of but not limited to demineralized bone matrix, freezedried and fresh ground soft tissue, such as submucosa, fascia, muscle,dermis, cartilage, or amnionic membrane among others. The microparticlescan also be microbeads made of biocompatible natural or syntheticpolymers, such as collagen, chitosan, alginate, agarose, or hyaluronicacid. The microparticles can also be conjugated with cytokines orbioactive growth supplements. The cytokines may be one or more of, forexample, IL-1αR antibody, TNF-a receptor antagonist, cyclooxygenase-2specific inhibitors, MAP kinase inhibitors, NO synthase inhibitors,NF-κB inhibitors, and inhibitors of MMP. The bioactive growthsupplements may be, for example, natural or recombinant FGF-family,TGF-family, IGF-1, PDGF, EGF, VEGF, HGF, PTHrP, Ihh, dexamethasone,insulin, transferrin, selenium, ITS, or ascorbate. The bioactive growthsupplements may also be, for example, factors extracted fromdemineralized bone matrix, basement membrane, or submucosa matrix.

If desired, the circumferential surface and/or superior aspect of thecartilage part of the graft can be microperforated using enzyme linkedmicroparticles as described in U.S. Pat. Nos. 6,432,712 and 6,416,995.These patents are hereby incorporated by reference in their entireties.The size of the microparticles may range from about 20 to about 500micrometer. Alternatively, the microperforation can be conducted bymechanical or laser drilling on the cartilage such that holes ofapproximately 20 to 500 micrometer in diameter may be created. Themicroperforation can be conducted before or after the cleaning,disinfection, devitalization process.

FIG. 7-FIG. 12 illustrate the tools used for crafting the osteochondralplugs or cartilage discs or slices. FIG. 7 demonstrates a holder (63)designed to secure an osteochondral plug (5) or (20) or (28) duringcrafting to obtain osteochondral plugs (8 a), (8 b), (10), and (14); or(22 a), (22 b), (23), and (25); or (30 a), (31), respectively. The innerdiameter of the cylindrical holder may be slightly larger than thelargest diameter of the osteochondral plug. Slots (64) illustrated inFIG. 7(a) may be created along the longitudinal direction of the hollowcylindrical holder according to the width, the length, the amount andthe orientation of the gaps (such as gap 9) to be created on theosteochondral plug. The inner surface of the bottom portion of theholder (63) may be threaded (59) so that a custom made bolt (60) can bethreaded into to support the osteochondral plug along the longitudinaldirection during crafting as illustrated in FIGS. 7(d, e, and f). Theouter surface of the bottom portion of the holder (63) may be flattened(58) and made rough so that the holder (63) can be fit into a lathe or aclamp of a drilling and/or milling machine during crafting. The clampcan be fixed on a table of the drilling/milling machine to enablemovement in multiple directions. The table can also move bothperpendicular to and parallel to the spindle axis of the endmill ordrill bit to accomplish cutting. When the osteochondral plug may beinserted in the holder, the cartilage cap may be positioned to face downand supported by the custom made bolt (60) as illustrated in FIGS. 7(d,e, and f). Then, in this aspect, set screws (57), preferably to beoriented 90 degrees apart, may be engaged to further secure theosteochondral plug within the holder (63) and to adjust the centerlineof the osteochondral plug to be parallel to the cutting tool centerlineor cutting direction. The set screws (57) can be oriented parallel to orat an angle relative to the articular surface of the osteochondral plugas illustrated in FIGS. 7(d and e). The angular orientation of the setscrew(s) can provide extra support on the osteochondral plug duringcrafting to minimize the stress exerting on the cartilage cap. Thecrafting can be conducted by sawing, or drilling and/or milling from thetop, i.e., the bottom of the osteochondral bone portion. FIG. 8demonstrates a holder (61) designed to secure an osteochondral plug (5)or (20) or (28) during crafting to obtain osteochondral plugs (12) or(24) or (32), respectively. The inner diameter of the cylindrical holdermay be slightly larger than the largest diameter of the osteochondralplug. Slots (62) illustrated in FIGS. 8(a and b) may be created alongthe longitudinal direction of the hollow cylindrical holder according tothe diameter and the amount and the orientation of the channels (13)created on the osteochondral plug. The inner surface of the bottomportion of the holder (61) can be threaded (59) so that a custom madebolt (60) can be threaded into to support the osteochondral plug alongthe longitudinal direction during crafting as illustrated in FIGS. 8(d,e, and f). The outer surface of the bottom portion of the holder (61)may be flattened (58) and made rough so that the holder (61) can be fitinto a clamp during crafting. When the osteochondral is inserted in theholder, the cartilage cap may be positioned to face up and the boneportion may be supported by the custom made bolt (60) as illustrated inFIGS. 8(d, e, and f). Then, in this aspect, set screws (57), preferablyto be oriented 90 degrees apart, may be engaged to further secure theosteochondral plug within the holder (61) and to adjust the superficialsurface of the cartilage cap on the osteochondral plug such that it maybe parallel to the bottom surface of the custom made bolt (60). The setscrews (57) can be oriented parallel to or at an angle relative to thearticular surface of the osteochondral plug as illustrated in FIGS. 8(dand e). The angular orientation of the set screw(s) can provide extrasupport on the osteochondral plug during crafting by forcing the boneportion of the osteochondral graft against the custom made bolt (60).The crafting can be conducted by drilling and milling through the slots(62) towards the circumferential surface of the bone portion of theosteochondral grafts. FIG. 9 demonstrates a holder (54) designed tosecure an osteochondral plug (5) or (20) or (28) during crafting toobtain osteochondral plugs (16) and (18); or (26) and (27); or (34) and(35), respectively. The inner diameter of the cylindrical holder may beslightly larger than the largest diameter of the osteochondral plug.Slots (56) illustrated in FIGS. 9(a and b) may be created along thecircumferential direction of the hollow cylindrical holder according tothe diameter and the amount and the orientation of the channels (17) orslots (19) to be created on the osteochondral plug. The inner surface ofthe bottom portion of the holder (54) may be threaded (59) so that acustom made bolt (60) can be threaded into to support the osteochondralplug along the longitudinal direction during crafting as illustrated inFIGS. 9(d, e, and f). The outer surface of the bottom portion of theholder (54) may be flattened (58) and made rough so that the holder (54)can be fit into a clamp to facilitate gripping during crafting. When theosteochondral plug is inserted in the holder, the cartilage cap may bepositioned to face up and the bone portion may be supported by thecustom made bolt (60) as illustrated in FIGS. 9(d, e, and f). Then, inthis aspect, set screws (57), preferably to be oriented 90 degreesapart, may be engaged to further secure the osteochondral plug withinthe holder (54) and to adjust the superficial surface of the cartilagecap on the osteochondral plug to be parallel to the bottom surface ofthe custom made bolt (60). The set screws (57) can be oriented parallelto or at an angle relative to the articular surface of the osteochondralplug as illustrated in FIGS. 9(d and e). The angular orientation of theset screw(s) can provide extra support on the osteochondral plug duringcrafting by forcing the bone portion of the osteochondral graft againstthe custom made bolt (60). The crafting can be conducted by drilling andmilling through the slots (56) towards the circumferential surface ofthe bone portion of the osteochondral grafts.

The shaped cartilage grafts can be further cleaned and disinfected.Examples of cleaning solutions and cleaning and disinfection methods aredescribed in U.S. Pat. Nos. 5,556,379, 5,820,581, 5,976,104, 5,977,034,5,977,432, 5,797,871, and 6,024,735. These patents are herebyincorporated by reference in their entireties.

For the cleaning process, the crafted osteochondral plugs can be placedinto a processing chamber (75) shown in FIG. 13a such that theosteochondral bone portion with or without gaps or a bore or channels orslots described above may be tightly fit into the cylindrical step holesin an insert (80). The insert (80) as illustrated in FIG. 13b canincorporate multiple osteochondral plugs, cartilage discs, or slices andhas a rubber ring (82) to create a seal between the wall of theprocessing chamber and the insert. The diameter of the top portion (84)of the step cylindrical hole (83) in the insert (80) is slightly largerthan the diameter of the cartilage portion on the osteochondral plug. Aporous ring (85), made of a porous material such as porous titanium,stainless steel, ceramics, hydroxyapatite, calcium phosphate, or calciumsulfate, with a center hole diameter slightly larger than the bottomportion (86) of the step cylindrical hole (83) can be fit in the topportion (84). The diameter of the bottom portion (86) of the stepcylindrical hole (83) may be slightly larger than the osteochondral boneportion of the osteochondral plug. A rubber ring (89) may be fitted inthe bottom portion of the step cylindrical hole (83). When any one ofthe osteochondral plugs in FIG. 2-FIG. 5 is fitted into the stepcylindrical hole (83), the inferior surface facing the osteochondralbone portion of the cartilage cap (6, 37, 39, 41, 43, or 45) may beplaced against the top surface of the porous ring (85) as illustrated inFIG. 13a . The bone portion can be fit into the bottom part (86) of thecylindrical hole (83) with the rubber ring (89) on the peripheralsurface that creates a seal. The cleaning solution (90), i.e., AlloWash®Solution (LifeNet, Inc., Virginia Beach, Va.), may be added from the topof the processing chamber. Under centrifugal force, preferably fromabout 100 to about 2000 rcf, more preferably from about 500 to about1500 rcf, most preferably from about 1000 to about 1400 rcf, thecleaning solutions can be induced to migrate through the tissues andinto the bottom of the processing chamber. Optionally, sonication can beconducted preferably for about 5 minutes to about 24 hours, morepreferably for about 0.5 to about 12 hours, and at frequency ofpreferably from 1 Hz to about 200 Hz, more preferably from 50 Hz toabout 100 Hz before the centrifugation process using an ultrasoniccleaner. Alternatively, the cleaning process can be conducted bycombining optional sonication and vacuum pressure (FIG. 14). Thecleaning solution (90 and 93), i.e., AlloWash® Solution, can be addedinto the processing chamber to have the entire graft submerged. Thegrafts can be optionally sonicated preferably for about 5 minutes toabout 24 hours, more preferably for about 0.5 to about 12 hours, and atfrequency of preferably from 1 Hz to about 200 Hz, more preferably from50 Hz to about 100 Hz. Then the grafts can be subjected to negativepressure from the bottom port (78), collection beaker (94), and the pump(95). After centrifugation or vacuuming, the waste (91) may be discardedand the osteochondral plugs may be removed from their respectiveprocessing chambers and the surface aspects of the plugs may be flushedusing pulsatile lavage with AlloWash® Solution, and optionally isotonicsaline to remove residual AlloWash® Solution from the grafts.

After the cleaning and disinfecting process, osteochondral plugs orcartilage discs or slices or flakes or curls can be placed in aprocessing chamber and devitalized using, for example, one of thefollowing methods: agitating on a shaker or rocker or mixer, or usingcentrifugal force (FIG. 13a ), or using vacuum pressure (FIG. 14), orusing a flow through system (FIG. 15), or using cyclic hydrodynamicpressure (FIG. 17a ). United States patents directed toward thedecellularization and/or devitalization of tissue, include U.S. Pat.Nos. 6,743,574, 6,734,018, 6,432,712, 6,416,995 and U.S. Patentapplication numbers 2004/0076657, 2004/0067582, and 2003/0219417. Thesepatents and patent applications are incorporated by reference in theirentireties.

After cartilage grafts are properly placed in the processing chamber ortubes, the cartilage grafts of the osteochondral plugs or discs orslices are optionally modified in a pretreatment solution. Thepretreatment solution may be composed of about 0.1 to about 10 U/mlenzymes, such as chondroitinase ABC in a buffer, such as Tris/NaAc amongothers. The pretreatment step can be conducted, for example, on a shakeror rocker or mixer, or in a processing chamber (75 or 96) under arelative centrifugal force, or under a vacuum pressure less than theambient pressure, or in a pressure induced flow through system, or undercyclic hydrodynamic pressure. By varying the duration of thepretreatment and the concentration of the chondroitinase ABC in thepretreatment solution, the amount of proteoglycan to be removed can becontrolled. Following completion of the pretreatment, the pretreatmentsolution may be removed from the tubes or the processing chamber (75 or96) and may be replaced with a rinsing solution. The cartilage graftscan be rinsed in the rinsing solution, such as water, saline, phosphatebuffer saline, RPMI media, balanced Hank's solution, Lactated Ringer'ssolution, DMEM/F12, F12, or DMEM media, among others, in thecorresponding processing chamber or tubes. The rinsing solution may bethen replaced with an extracting solution (Buffer, sodium dodecylsulfateor N-lauroyl sarcosinate or CHAPS, and BENZONASE® among others) withdecontaminating agents to disinfect the tissues and to digest thenucleic acids present in the plugs. The grafts can be incubated in atest tube that fits onto a shaker or rocker or mixer, or in a processingchamber (75 or 96) under a relative centrifugal force, or under vacuumpressure, or in a flow through system, or under cyclic hydrodynamicpressure to induce a fluid flow through the tissue to be devitalized asillustrated in FIG. 13-FIG. 17. Following completion of thedevitalization, the extracting solution may be removed from the tubes orthe processing chamber (75 or 96) and may be replaced by a rinsingsolution, such as water, saline, phosphate buffer saline, RPMI media,balanced Hank's solution, Lactated Ringer's solution, DMEM/F12, F12, orDMEM media, among others. The grafts can be incubated again in a testtube that fits onto a shaker or rocker or mixer, or in a processingchamber (75 or 96) under a relative centrifugal force, or under vacuumpressure, or in a flow through system, or under cyclic hydrodynamicpressure to induce a fluid flow through the tissue to be devitalized.

For devitalization under agitation, osteochondral plugs or cartilagediscs or slices or flakes or curls can be placed in one or multiple testtubes that may be fixed on a shaker or rocker or mixer. Cartilage graftscan be incubated with a pretreatment solution on preferably at atemperature from about 4° C. to about 45° C., more preferably from about15° C. to about 37° C., for a period of time preferably of about 1 toabout 24 hours, more preferably of about 1 to about 16 hours, and underagitation preferably of about 10 to about 1000 rpm, more preferably ofabout 100 to about 500 rpm. Cartilage grafts can be washed with isotonicsaline solution preferably at a temperature from about 4° C. to about42° C., more preferably from about 15° C. to about 37° C., for a periodof time preferably of about 10 minutes to about 24 hours, morepreferably of about 15 to about 60 minutes, and under agitationpreferably of about 10 to about 1000 rpm, more preferably of about 100to about 500 rpm. After washing with saline two more times, the isotonicsaline solution may be replaced by the extracting solution. The testtubes containing cartilage grafts can be incubated preferably at atemperature from about 4° C. to about 45° C., more preferably from about15° C. to about 37° C., for a period of time preferably of about 1 toabout 24 hours, more preferably of about 1 to about 16 hours, and underagitation preferably of about 10 to about 1000 rpm, more preferably ofabout 100 to about 500 rpm. Following completion of the devitalizationprocess, the tubes may be drained of the extracting solution andreplaced with a rinsing solution. The cartilage grafts can be washed inthe rinsing solution preferably at a temperature from about 4° C. toabout 45° C., more preferably from about 15° C. to about 37° C., for aperiod of time preferably of about 10 minutes to about 24 hours, morepreferably of about 15 to about 60 minutes, and under agitationpreferably of about 10 to about 1000 rpm, more preferably of about 100to about 500 rpm. The washing can be repeated for two more times. Thetubes may be then drained of the rinsing solution and replaced with astorage solution. The cartilage grafts can again be incubated onpreferably at a temperature from about 4° C. to about 42° C., morepreferably from about 15° C. to about 37° C., for a period of timepreferably of about 1 to about 24 hours, more preferably of about 1 toabout 16 hours, and under agitation preferably of about 10 to about 1000rpm, more preferably of about 100 to about 500 rpm.

For devitalization under centrifugal force, osteochondral plugs can befit into the cylindrical step holes in an insert (80 in FIG. 13a ) asdescribed in the cleaning process. Cartilage discs or slices can beplaced on a porous ring (85) in the top portion (84) of the stepcylindrical hole (83) in the insert (80). The insert can be made ofbiocompatible polymers such as Teflon, biocompatible metal such astitanium or stainless steel. The pretreatment solution may betransferred into the top part of the chamber. The chamber can becentrifuged preferably at a temperature from about 4° C. to about 45°C., more preferably from about 15° C. to about 37° C., for a period oftime preferably of about 10 minutes to about 24 hours, more preferablyof about 30 minutes to about 18 hours, most preferably of about 1 hourto about 16 hours, and at a speed preferably of from about 10 to about2000 rcf, more preferably of about 100 to about 1.500 rcf, mostpreferably of about 500 to about 1000 rcf. The pretreatment solution inboth the top and the bottom portion of the chamber may be removed andthe bottom cap (79) may be closed. Then the rinsing solution may betransferred into the top portion of the processing chamber. The chambercan be centrifuged preferably at a temperature from about 4° C. to about45° C., more preferably from about 15° C. to about 37° C., for a periodof time preferably of about 10 minutes to about 24 hours, morepreferably of about 30 minutes to about 18 hours, most preferably ofabout 1 hour to about 16 hours, and at a speed preferably of from about10 to about 2000 rcf, more preferably of about 100 to about 1500 rcf,most preferably of about 500 to about 1000 rcf. The washing can beoptionally repeated and the rinsing solution may be drained. Theextracting solution may be then transferred into the top portion of theprocessing chamber (FIG. 13a ). The processing chamber containingcartilage grafts can be centrifuged preferably at a temperature fromabout 4° C. to about 45° C., more preferably from about 15° C. to about37° C., for a period of time preferably of about 10 minutes to about 24hours, more preferably of about 30 minutes to about 18 hours, mostpreferably of about 1 hour to about 16 hours, and at a speed preferablyof from about 10 to about 2000 rcf, more preferably of about 100 toabout 1500 rcf, most preferably of about 500 to about 1000 rcf, tofacilitate penetration of the fluid into the cartilage graft. Followingcompletion of the devitalization process, the processing chamber may bedrained of extracting solution and replaced with a rinsing solution,such as water, saline, phosphate buffer saline, RPMI media, balancedHank's solution, Lactated Ringer's solution, DMEM/F12, F12, or DMEMmedia. The chamber can be centrifuged preferably at a temperature fromabout 4° C. to about 45° C., more preferably from about 15° C. to about37° C., for a period of time preferably of about 10 minutes to about 24hours, more preferably of about 30 minutes to about 18 hours, mostpreferably about 1 hour to about 16 hours, and at a speed preferably offrom about 10 to about 2000 rcf, more preferably of about 100 to about1500 rcf, most preferably of about 500 to about 1000 rcf. The washingcan be repeated twice and the rinsing solution may be drained. Therinsing solution may be replaced with a storage solution. The chambercan be centrifuged preferably at a temperature from about 4° C. to about45° C., more preferably from about 15° C. to about 37° C., for a periodof time preferably of about 10 minutes to about 24 hours, morepreferably of about 30 minutes to about 18 hours, most preferably ofabout 1 hour to about 16 hours, and at a speed preferably of from about10 to about 2000 rcf, more preferably of about 100 to about 1500 rcf,most preferably of about 500 to about 1000 rcf.

For devitalization in a fluid through system (FIG. 15 or FIG. 16a ),osteochondral plugs, cartilage discs, cartilage slices, or cartilageflakes or curls can be loosely fit into an insert (101) that may be madeof a porous material, such as porous titanium, stainless steel, orceramics (FIG. 15). Fluid may be allowed to flow through and around thecartilage grafts. The superficial surface of the cartilage grafts can beperpendicular to the fluid flow directions as illustrated in FIG. 15.Alternatively, the cartilage grafts can be placed in an insert (274)with a porous plate (275) (FIG. 16a and FIG. 16b ). The porous plate(275), made of a porous material, such as porous titanium, stainlesssteel, or ceramics, has slots that allow cartilage portion of the graftsto be fit into so that the fluid flow may be parallel to the superficialsurface of the cartilage graft as illustrated in FIG. 16 a.

In detail, FIG. 15 and FIG. 16a illustrate a system for processingcartilage grafts using a flow through system to circulate thepretreatment, extracting, rinsing, or storage solution between theprocessing chamber and the corresponding reservoir. The reservoir (103)can be interchangeably a pretreatment solution reservoir and anextracting solution reservoir. Moreover, the reservoir (104) can beinterchangeably a rinsing solution reservoir or a storage solutionreservoir. Cartilage grafts may be placed into the processing chamber(96) using a suitable insert (101 or 274) made of porous polymer, metalor ceramics. The insert (101 or 274), shown in FIG. 15 or FIG. 16a , canaccommodate multiple grafts. The Luer lock (92) and the lid (97) may bescrewed down tightly to engage the o-ring thereby eliminating leakagefrom the chamber (96). The hydrophobic adsorbent resin and anionexchange resin are optionally added to the resin chamber (102). Theremay be an o-ring at the top and bottom of the resin chamber to ensure asecure fit between the resin chamber and the resin housing to force theflow of rinsing solution through the resin chamber. Sterile medicalgrade disposable tubing may be attached to ports (110, 108, 78, 112,111, 107, 105, 98), and with 3-way stop cocks (113, 114, 115, and 116)inserted in-line. The tubing may be attached to the sipper devices (106and 109) such that the return flow enters the side with the shortestspout and the outbound flow may be pulled through the longest spout. Thetubing may be placed onto the rollers of the peristaltic pumps (95 and117) and the clamp lowered to hold the tubing in place. Once the rinsingor storage solution (104), pretreatment or the extracting solution (103)may be connected, all connections may be checked to ensure that they aretight. The pumps (95 and 117) may be turned on and their calibration ispreferably checked. The pretreatment solution or the extracting solutionmay be drawn up from the long spout of sipper (106), proceeds throughthe port (105), continues past stopcocks (113) and tubing through theroller assembly of the pump (95) into the processing chamber (96)through port (98), proceeds through the cartilage graft and insert, thenout the bottom of the chamber and through port (78) and continues paststopcocks (114 and 115), then into the sipper (106) through the shortspout and port (107) by using a second pump (117). This cycle can becarried out at a flow rate preferably of from about 2 mls/minute toabout 500 mls/minute, more preferably of from about 50 mls/minute toabout 350 mls/minute, most preferably of from 150 mls/minute to about250 mls/minute, at a temperature preferably of about 4 to about 45° C.,more preferably of about 15 to about 37° C., and a period of timepreferably of from about 1 hour to about 48 hours, more preferably offrom about 1 hour to about 24 hours, and most preferably of from about 1hour to about 16 hours. After the pretreatment and/or extraction, thepump (95) may be stopped and only pump (117) may be on until theprocessing chamber is empty. Stopcocks (113, 114, 115, and 116) may beturned to redirect the flow to and from the rinsing solution reservoir(104) and to optionally direct the flow through the resin housingchamber (102). The pumps (95 and 117) may be turned on again and thechamber may be filled by the rinsing solution, exiting sipper (108) outthe long spout, into the tubing through stopcock (113), and through theroller pump (95), through the processing chamber (96) into the tissuechamber through port (98) and proceeds through the cartilage graft andinsert, then out the bottom of the chamber and through port (78) andcontinues past stopcock (114) which directs the flow of the rinsingsolution into the resin chamber (102) out port (111) and stopcocks (115and 116) through the tubing and into sipper (109) via the short spoutand port (110) and into the isotonic saline or water reservoir (104) byusing a second pump (117). This washing cycle can be carried out at aflow rate preferably of from about 2 mls/minute to about 500 mls/minute,more preferably of from about 50 mls/minute to about 350 mls/minute,most preferably of from 150 mls/minute to about 250 mls/minute, at atemperature preferably of about 4 to about 45° C., more preferably ofabout 15 to about 37° C., and a period of time preferably of from about1 hour to about 48 hours, more preferably of from about 1 hour to about24 hours, and most preferably of from about 1 hour to about 16 hours.The pressure within the processing chamber can be monitored by apressure gauge (100) that may be connected to a port (99). Then therinsing solution in reservoir (104) may be replaced by a storagesolution and the circulation can be carried out at a flow ratepreferably of from about 2 mls/minute to about 500 mls/minute, morepreferably of from about 50 mls/minute to about 350 mls/minute, mostpreferably of from 10 mls/minute to about 50 mls/minute, at atemperature preferably of about 4 to about 45° C., more preferably ofabout 15 to about 37° C., and a period of time preferably of from about1 hour to about 48 hours, more preferably of from about 1 hour to about24 hours, and most preferably of from about 1 hour to about 16 hours.

For devitalization under cyclic hydrodynamic pressure (FIG. 17a ),cartilage grafts may be placed into the processing chamber (96) using asuitable insert (118), shown in FIG. 17b made of biocompatible polymerssuch as Teflon, or biocompatible metal such as titanium or stainlesssteel. The insert (118) can accommodate multiple grafts with differentconfiguration, such as a stack of cartilage slices, cartilage disc, orosteochondral plug as illustrated in FIG. 17a . If desired, acylindrical well (124) that has thread on the half of the outer surfacemay be threaded onto the top portion (120) of a step cylindrical hole(119). A porous platen (129) and an o-ring (130) may be fittedunderneath of the well (124) so that fluid flow (if present) may be onlyallowed to go through the middle of the well (124). The cartilage discsor slices or cartilage flakes can be placed on the porous platen (129)within the well (124) as illustrated in FIG. 17a . The insert (118) andthe well (124) may be made of the same material as the insert (80). Ifdesired, the cartilage discs or thin slices of cartilage stackedtogether, as illustrated in FIG. 17a , can be placed between twocontoured porous platens, which create curvature match the defect sitein a joint. The hydrodynamic cyclic pressure can be driven by compressedair/gas to pressurize the pretreatment solution or the extractingsolution or rinsing solution or storage solution within the processingchamber (96) to facilitate the processing. The Luer lock (92) and thelid (97) may be screwed down tightly to engage the o-ring therebyeliminating leakage from the chamber (96). The processing chamber can befilled with a processing solution. A pressurization chamber, composed ofthe bottom (282) and the top (285) parts, may be threaded together andseparated by a fluid impermeable flexible membrane (284) and sealed byan o-ring (283). The pressurization chamber may be connected to anair/gas chamber (133) and a piston (132) through a connector (286). Thebottom of the pressurization chamber may be filled with processingsolution and connected with the processing chamber (96) through port(287) and rigid tubing. The compressed air/gas can be driven by a piston(132) and passes through the connector (286) to compress the flexiblemembrane (193). The piston can be driven by a computer controlled camand/or stepper motor to move up and down to create a cyclic pressure onthe flexible membrane that transfers the pressure to the processingchamber. The pressure can be monitored using two pressure gauges (100)and regulated by two valves (131), which may be connected to the rigidtubing. The compressed air/gas may be made of sterile 5% CO₂ in air.

During devitalization, the pretreatment solution may be transferred intothe processing chamber, as well as the rigid tubing and the bottom partof the pressurization chamber (FIG. 17a ). The cartilage grafts may bepre-treated with pretreatment solution under cycles of hydrodynamicpressure preferably of about −20 to about 20 MPa, more preferably about−10 and about 10 MPa, most preferably about −6 and about 6 MPa, at afrequency preferably of from about 0.01 to about 5 Hz, more preferablyof from about 0.1 to about 2 Hz, and most preferably of from about 0.5to about 1 Hz, at a temperature preferably of from about 4 to about 45°C., more preferably of from about 15 to about 37° C., and for a periodof time preferably of from about 5 minutes to about 48 hours, morepreferably of from 10 minutes to about 24 hours, most preferably of fromabout 30 minutes to about 16 hours The pretreatment solution in theprocessing chamber may be removed and replaced by a rinsing solution.The grafts can be pressurized again under cycles of hydrodynamicpressure preferably of about −20 to about 20 MPa, more preferably about−10 and about 10 MPa, most preferably about −6 and about 6 MPa, at afrequency preferably of from about 0.01 to about 5 Hz, more preferablyof from about 0.1 to about 2 Hz, and most preferably of from about 0.5to about 1 Hz, at a temperature preferably of from about 4 to about 45°C., more preferably of from about 15 to about 37° C., and for a periodof time preferably of from about 5 minutes to about 48 hours, morepreferably of from 10 minutes to about 24 hours, most preferably of fromabout 30 minutes to about 16 hours. After rinsing solution may bedrained from the processing chamber, an extracting solution may betransferred into the processing chamber. The cartilage grafts can beprocessed under cycles of hydrodynamic pressure preferably of about −20to about 20 MPa, more preferably about −10 and about 10 MPa, mostpreferably about −6 and about 6 MPa, at a frequency preferably of fromabout 0.01 to about 5 Hz, more preferably of from about 0.1 to about 2Hz, and most preferably of from about 0.5 to about 1 Hz, at atemperature preferably of from about 4 to about 45° C., more preferablyof from about 15 to about 37° C., and for a period of time preferably offrom about 5 minutes to about 48 hours, more preferably of from 10minutes to about 24 hours, most preferably of from about 30 minutes toabout 16 hours. Following completion of the devitalization process, theprocessing chamber may be drained of the extracting solution andreplaced with rinsing solution, such as water, saline, phosphate buffersaline, RPMI media, balanced Hank's solution, Lactated Ringer'ssolution, DMEM/F12, F12, or DMEM media. The cartilage grafts can bepressurized again under cycles of hydrodynamic pressure preferably ofabout −20 to about 20 MPa, more preferably about −10 and about 10 MPa,most preferably about −6 and about 6 MPa, at a frequency preferably offrom about 0.01 to about 5 Hz, more preferably of from about 0.1 toabout 2 Hz, and most preferably of from about 0.5 to about 1 Hz, at atemperature preferably of from about 4 to about 45° C., more preferablyof from about 15 to about 37° C., and for a period of time preferably offrom about 5 minutes to about 48 hours, more preferably of from 10minutes to about 24 hours, most preferably of from about 30 minutes toabout 16 hours. The rinsing solution may be replaced with a storagesolution. The cartilage grafts can be pressurized again under cycles ofhydrodynamic pressure preferably of about −20 to about 20 MPa, morepreferably about −10 and about 10 MPa, most preferably about −6 andabout 6 MPa, at a frequency preferably of from about 0.01 to about 5 Hz,more preferably of from about 0.1 to about 2 Hz, and most preferably offrom about 0.5 to about 1 Hz, at a temperature preferably of from about4 to about 45° C., more preferably of from about 15 to about 37° C., andfor a period of time preferably of from about 5 minutes to about 48hours, more preferably of from 10 minutes to about 24 hours, mostpreferably of from about 30 minutes to about 16 hours.

All the inserts (80, 101, 274, and 118) described above may be designedto be interchangeable among all the processing chambers (75 or 96) inall the devitalization methods. Osteochondral plugs or cartilage discsor slices or flakes or curls from the same donor can be fit into asingle processing chamber.

If desired, as described above, after devitalization, thecircumferential area of the cartilage graft, such as the cartilageportion of the osteochondral plug, or cartilage discs, or cartilageslices may be further crafted to maximize the surface and contact areasbetween the boundaries of the recipient cartilage being repaired and thecartilage graft, as illustrated in FIG. 5, to facilitate integration ofthe graft tissue to the recipient tissue.

The cartilage grafts that have been crafted and devitalized as notedabove can be stored in a plasticizer, such as 15-77% glycerol. Suitablestorage solutions are well known to those of ordinary skill in the artto which the present invention applies, and such solutions may bereadily selected and employed by those of ordinary skill in the art towhich the present invention applies without undue experimentation. U.S.Pat. Nos. 6,544,289, 6,293,970, 6,569,200, and 7,063,726 directed towardthe use of a water replacing agent for storage of bone and soft tissue.These patents are incorporated by reference in their entireties. Aftercompletion of the incubation with storage solution, in one embodiment,the cartilage grafts can be placed in an inner bottle (134) of varyingsize to accommodate a small (a) or a large graft (b) and completelyimmersed in the storage solution (FIG. 18a ). The lid (136) of the innerbottle may be screwed down tightly to engage an o-ring therebyeliminating leakage from the bottle. The two ports (137 and 138) sealedwith Luer lock caps on the lid (136) can be used for a future rinsingstep in the operating room. The inner bottle may be then placed in anouter container (139) made of foam material that functions as a cushionif impact force applies. The entire package can be sealed with a lid(140). Alternatively, storage solution soaked grafts can be spun quicklyto remove excessive storage fluid and packaged in double containers asillustrated in (c-e) in FIG. 18b . The cartilage grafts can be placed inan inner sealed box (141) then the inner box may be placed in an outerbox (143) and sealed (c in FIG. 18b ); or placed in an inner bag (145)with two ports (147), sealed under vacuum on one edge (146), placed inan outer bag, and sealed (d and e in FIG. 18b ). Depending on the sizeof the grafts, the inner bag (145) can be large enough to accommodate awhole condyle as illustrated in (d in FIG. 18b ) or small enough to fitan osteochondral plug as illustrated in (e in FIG. 18b ). The two ports(147) sealed with Luer lock caps (148) on the sealed edge (146) can beused for a future rinse step in the operating room. The grafts in thestorage containers described above may be terminally sterilized usingmethods known in the art including, but not limited to, gammairradiation. Alternatively, the devitalized cartilage grafts may beterminally sterilized with super critical CO₂ or ethylene oxide beforesoaked in the sterile storage solution and packaged in a sterile field.

The devitalized cartilage grafts as shown in FIG. 1-FIG. 6 can beoptionally modified to stimulate in vivo, in situ, and/or in vitroinfiltration of the viable cells, such as chondrocytes from cartilagetissue or stromal cells from bone marrow or synovium. In one embodiment,after devitalization and washing, the cartilage graft can be coated withone or more agent(s) that has bioactive growth supplement or cytokinebinding site(s) through covalent coupling or adsorption to increase theaffinity of a bioactive growth supplement or cytokine to the devitalizedgraft. The agent(s) that has bioactive growth supplement or cytokinebinding site(s) can be one or a combination of extracellular matrixproteins. The agent(s) that has bioactive growth supplement or cytokinebinding site(s) can be a natural or synthetic molecule. Moreover, theagent may comprise an extra functional moiety that may be selected froma group, but not limited to, COOH, NH2, or OH can be added to thenatural or synthetic proteins or peptides to facilitate the coating. Theextra functional moeity can be from groups that change thehydrophilicity or charge. After being coated with one or more agent(s)that has bioactive growth supplement or cytokine binding site(s), thecartilage graft as a whole unit can be further soaked with one or morebioactive growth supplements or cytokines. The cartilage portion and thebone portion (if present) of a cartilage graft can be treated at thesame time with the same bioactive growth supplements. Alternatively, thecartilage portion and the bone portion of a cartilage graft can betreated separately, e.g., the cartilage portion may be soaked into oneor more than one chondrogenic factor(s) and the bone portion may besoaked into one or more than one osteogenic factor(s). In addition, inorder to facilitate the binding of the bioactive growth supplement orcytokines to the cartilage graft, a solution with one bioactive growthsupplement or cytokines, or a cocktail of bioactive growth supplementsor cytokines can be added into the top and/or bottom portion of theprocessing chamber (75 or 96). Under centrifugal force, or vacuumpressure, or a pressure induced fluid flow, or a cyclic pressurization,the bioactive growth supplements or cytokines can be induced to migrateinto the cartilage graft. Alternatively, microparticles can also beconjugated with a bioactive growth supplement or a cytokine and forcedinto the devitalized cartilage using centrifugal forces between 50 and2000 rcf, preferably between 100 and 1800 rcf, and more preferablybetween 500 and 1500 rcf. The microparticles can be from a group of, butnot limited to, demineralized bone matrix, freeze dried and ground softtissue, such as submucosa, fascia, muscle, dermis, cartilage, or amionicmembrane. The microparticles can also be microbeads made ofbiocompatible natural or synthetic polymers, such as collagen, chitosan,alginate, agarose, or hyaluronic acid. The bioactive growth supplementsmay be one or more of, a natural or recombinant FGF-family, TGF-family,IGF-1, PDGF, EGF, VEGF, HGF, PTHrP, Ihh, dexamethasone, insulin,transferrin, selenium, ITS, or ascorbate. The bioactive growthsupplements can also be from the extractions of demineralized bonematrix, basement membrane, or submucosa matrix. The cytokines may be oneor more of, for example, an IL-1αR antibody, TNF-a receptor antagonist,cyclooxygenase-2 specific inhibitors, MAP kinase inhibitors, NO synthaseinhibitors, NF-κB inhibitors, and inhibitors of MMP.

The devitalized cartilage graft is intended to be recellularized insitu, in vitro, or in vivo. The devitalized cartilage graft can beremoved from the storage container, rinsed, and diluted using anAlloFlow™ chamber among others. Such a chamber is disclosed in U.S. Pat.Nos. 5,879,876 and 6,326,188, which are incorporated by reference intheir entireties herein. In one embodiment, the devitalized cartilagegraft can be recellularized in situ. The devitalized cartilage graft canbe implanted in a cartilage defect in a recipient to render cells fromthe recipient tissue to migrate into the devitalized cartilage graft,proliferate, differentiate, and secrete endogenous extracellular matrix.In order to facilitate the in situ recellularization, chemical stimulican be optionally applied. The chemical stimuli can be to coat adevitalized cartilage graft with one or more agent(s) that havebioactive growth supplement or cytokine binding site(s) to increase theaffinity of chondrogenic and/or osteoinductive factor adsorption ontothe devitalized graft. The chemical stimuli can also be micro-particlesthat are conjugated with cytokines or bioactive growth supplements andsprayed or blasted onto the cartilage graft before implantation.Alternatively, for in situ recellularization, the devitalized grafts canbe recellularized by seeding recellularizable cells, for example, cellsisolated from autologous or allogenous soft tissue or bone marrow and/orcultured previously, on to the cartilage graft right beforeimplantation. FIG. 19 illustrated the procedure of renderingrecellularization of a cartilage disc or two parts of the cartilage discfrom superficial-mid or mid-deep region and implanting therecellularized cartilage grafts into the defect site. The cartilage disc(shown with star shape) with full depth (152) or cut from superficialand mid zone (152 a) or from mid and deep zone (152 b) along the depthmay be cleaned, disinfected, devitalized, and/or stored. Prior toimplantation, the cartilage discs may be rinsed with isotonic salineusing an AlloFlow™ chamber. Recellularizable cells isolated fromautologous or allogenous sources can be seeded on the devitalizedcartilage discs immediately before implantation. Optionally, acentrifugal force or a positive pressure can be applied to facilitatecell adhesion onto the devitalized cartilage graft. The devitalizedcartilage disc can be recellularized with one or more than one type ofcells from recellularizable cells. If desired, the superficial and midzone cartilage (152 a) can be seeded with chondrocytes from thesuperficial region, while the mid and deep zone cartilage (152 b) can beseeded with chondrocytes from the mid to deep region. During surgery,the blind bore (155) in the bone portion (156) of the cartilage defectcan be filled with a bone filler that may be a mixture of a matrix (157)with or without a carrier (158). U.S. Pat. No. 6,340,477, and U.S.patent application Ser. Nos. 11/247,230, 11/247,229, and 11/247,249,which are incorporated by reference in their entireties herein, aredirected towards the use of DBM combined with carriers that may behydrogel, synthetic or biological polymers to form a malleable boneputty or flowable gel for filling bone defects. The matrix in the bonefiller may be one or more of, for example, autologous crushed boneharvested from the defect site; demineralized bone matrix; cancellousand cortical bone mixture; small intestine submucosa, amniotic membrane,ligament, tendon, skin, muscle tissue, periostieum, or synovial tissue;ceramics; hydroxyapatite; calcium phosphate; calcium sulfate; poroussurgical grade titanium or stainless steel; or any combination of theabove. The matrix can be in the format of a sheet, a disc, a tape, asponge, a cube, a solid or hollow cylinder, gel, putty, or particles.The carrier may be one or more of, for example, dihydroxyphenylalanine(DOPA) based adhesive, glucose, concentrated albumin, cyanoacrylateadhesive, gelatin-resorcin-formalin adhesive, chondroitin sulfatealdehyde N-acetylglucosamine (GlcNAc), mussel-based adhesive, poly(aminoacid)-based adhesive, cellulose-based adhesive, synthetic acrylate-basedadhesives, platelet rich plasma (PRP), monostearoyl glycerolco-Succinate (MGSA), monostearoyl glycerol co-succinate/polyethyleneglycol (MGSAPEG) copolymers, or a combination comprising at least one ofthe foregoing polymers. The carrier can also be one or more of, forexample, native or modified collagen, gelatin, agarose, modifiedhyaluronic acid, fibrin, chitin, biotin, avidin, native or crosslinkedchitosan, alginate, demineralized bone matrix, MATRIGEL®, HUMANEXTRACELLULAR MATRIX®, homogenized connective tissue, proteoglycans,fibronectin, laminin, fibronectin, elastin, heparin, glycerol, or acombination comprising at least one of the foregoing polymers. Thecarrier may include bioactive growth supplements such as FGF-family,TGF-family, IGF-1, PDGF, EGF, VEGF, HGF, PTHrP, Ihh, dexamethasone,insulin, transferrin, selenium, ITS, or ascorbate. The carrier may alsoinclude bioactive growth supplements from the extractions ofdemineralized bone matrix, basement membrane, or submucosa matrix. Thecarrier may include cytokines, for example, an IL-1αR antibody, TNF-areceptor antagonist, cyclooxygenase-2 specific inhibitors, MAP kinaseinhibitors, NO synthase inhibitors, NF-κB inhibitors, or inhibitors ofMMP. Moreover, the carrier may also include one or more than one type ofcells from recellularizable cells. The bone filler may also be acortical and/or cancellous bone plug. After the blind bore (155) in thebone portion may be filled with a bone filler to provide a support, therecellularized cartilage disc, either full depth (153) or a stack ofcartilage slices from different zones (153 a and 153 b) to form a fulldepth cartilage (153 c), can be tight-fit into the blind bore (155) ofthe cartilage portion (154) of the defect site.

If the defect site that needs to be repaired has a curvature, thecartilage graft can be contoured to match the curvature. FIG. 20illustrates one of the methods to create a contoured graft in situ,wherein a cartilage disc (6) can be tailored into thin slices of varyingthickness and diameters (127 a-127 c), stacked, and implanted. Ifrecellularization is needed, cells can be seeded on the devitalizedcartilage slices in situ, i.e., immediately before stacking andimplantation. Alternatively, the cartilage discs and/or slices may berecellularized in vitro, i.e., seeded with cells, stacked, and culturedin a bioreactor to allow cell attachment, infiltration, proliferation,and/or differentiation. Nonetheless, the cartilage discs and/or slicescan be recellularized in vivo, i.e., implanted in soft tissue, such asmuscle pouch or fat pad or other tissue with progenitor or stromalcells, retrieved after about 7 days to about 3 month, and implanted. Thedevitalized cartilage slices can be recellularized with one or more thanone type of cells from recellularizable cells. If desired, thesuperficial and mid zone cartilage (152 a) can be seeded withchondrocytes from the superficial region, and the mid and deep zonecartilage (152 b) can be seeded with chondrocytes from the mid to deepregion. The cartilage slices can be optionally bonded between adjacentslices using one or more than one bonding agents. In one embodiment,during surgery, a step cylindrical osteochondral plug (30) with a flatsuperficial surface and gaps in the bone portion can be fit into theblind bore (155) first. The gaps or a bore or channels or slots in thebone portion of the osteochondral plug (30) can be filled with a bonefiller that may be a mixture of a matrix (157) with or without a carrier(158) as described in FIG. 19. The bone portion of the osteochondralplug can be tightly fit into the bone portion of the blind bore (155).Alternatively, the bone portion of the osteochondral plug can be looselyfit into the bone portion of the blind bore (155). The clearance betweenthe bone portion of the osteochondral plug and the blind bore (155) canbe filled with the same bone filler as in the gaps or a bore or channelsor slots on the bone portion of the osteochondral plug. The cartilageportion of the osteochondral plug can be tight-fit into the blind bore(155) of the convex cartilage portion (161) of the defect site. Ideally,the osteochondral plug fit in the defect site may be slightly lower thanthe surrounding recipient tissue so that the thin cartilage slices (127a-127 c) can be stacked on top of the osteochondral plug to match theoverall contour of the joint. Alternatively, during surgery, the blindbore (155) in the bone portion (156) of the cartilage defect can befilled with bone filler that may be a mixture of a matrix (157) with orwithout a carrier (158) as described in FIG. 19. The cartilage sliceswith (160) of without recellularization and with varying diameters canbe stacked and tight-fit into the cartilage portion (161) of the blindbore (155) at the defect site to match the overall contour of the joint.

In one embodiment, if desired, the cartilage matrix, such asosteochondral plugs, cartilage discs, slices, or flakes or curls can berecellularized in vitro and cultured optionally under chemical andmechanical stimuli for about 1 day to about 40 days to create a viablecoherent, contoured, and functional cartilage graft before implantation.The chemical stimuli during the in vitro recellularization andcultivation can be applied by adding one or a cocktail of bioactivegrowth supplements in the culture media. Alternatively, the chemicalstimuli can be applied by coating the devitalized cartilage with one ormore agent(s) that has bioactive growth supplement or cytokine bindingsite(s) through covalent coupling or adsorption to increase the affinityof a bioactive growth supplement or cytokine to the devitalized graft asillustrated previously. Furthermore, chemical stimuli can be applied bysprayed or blasted micro-particles onto the circumferential surface ofthe devitalized cartilage graft before recellularization. Themicroparticles may be, but are not limited to, demineralized boneparticles; or freeze dried and ground submucosa, fascia, muscle, dermis,or cartilage. The microparticles can also be microbeads made of naturalor synthetic materials that are conjugated with cytokines or bioactivegrowth supplements. The bioactive growth supplements may be one or moreof, for example, a natural or recombinant FGF-family, TGF-family, IGF-1,PDGF, EGF, VEGF, HGF, PTHrP, Ihh, dexamethasone, insulin, transferrin,selenium, ITS, or ascorbate. The bioactive growth supplements can alsobe from extractions of demineralized bone matrix, basement membrane, orsubmucosa matrix. The cytokines may be, but are not limited to, one ormore of, an IL-1.alpha.R antibody, TNF-a receptor antagonist,cyclooxygenase-2 specific inhibitors, MAP kinase inhibitors, NO synthaseinhibitors, NF-κB inhibitors, or inhibitors of MMP.

The mechanical stimulus may be applied using a bioreactor. Thecomponents of a bioreactor that can provide various modes of mechanicalstimuli are illustrated in FIG. 21 and FIG. 22. FIG. 21 illustrates thecomponents that can be assembled to become the bottom portion of achamber of a bioreactor for in vitro recellularization and cultivationof devitalized cartilage grafts. Three major components, i.e., thebottom cylindrical well (175), the cylindrical culture well (162), andthe cylindrical confining ring (204) can be assembled together tocomprise the bottom assembly (209). As illustrated, the top hole (177)of the bottom well (175) may be threaded so that the threaded outersurface (163) of the culture well (162) can be screwed into the top hole(177). A groove (181) may be also created at the bottom of the top hole(177) so that an o-ring (174) can be fit into. The culture well (162)may be screwed down into the top hole (177) of the bottom well (175) toengage the o-ring (174) thereby eliminating leakage from the chamber. Ifthe mechanical stimulus may be driven by a compressed air/gas asillustrated in FIG. 23, a gas permeable and water impermeable membrane(172) that can be fixed in a ring fixture (173) can be assembled betweenthe culture well (162) and the o-ring (174). The ports (179 and 180) onthe bottom well and the ports (170 and 171) on the culture well (162)can be used for either fluid or gas exchange, or media sample collectionduring culture. A confining ring (204) may be screwed down to thethreaded hole (164) of the culture well (162) to engage the porousplaten (206) and o-ring (207) thereby forcing the culture media ifpresent to flow though only in the middle of the confining ring duringmechanical simulation.

FIG. 22 illustrates the components that can be assembled to become thetop portion of the chamber of a bioreactor for in vitrorecellularization and cultivation of devitalized cartilage. Three majorcomponents, i.e., a cylindrical bushing (182), a cylindrical top cover(184), and a cylindrical top well (195) can be assembled together tocomprise the top assembly (211). The bushing (182) can be used as aguidance when a loading shaft (224) may be placed in the middle forconfined or unconfined compression tests as illustrated in FIG. 25, FIG.28, and FIG. 29. If the mechanical stimulation does not involve aloading shaft as illustrated in FIG. 23 and FIG. 24, the bushing (182)can be sealed with a cap (212). A groove (190) may be created at the topof the threaded hole (189) in the top cover (184) so that an o-ring(192) can be fit into. The top well (195) may be screwed into thethreaded hole (189) in the top cover (184) to engage the o-ring (192)thereby eliminating leakage from the chamber. If the mechanical stimulusmay be driven by a compressed air/gas as illustrated in FIG. 23, a gaspermeable and water impermeable membrane (193) that may be fixed in aring fixture (194) can be assembled between the top well (195) and theo-ring (192). The ports (198 and 199) on the top well (195) and the port(188) on top cover (184) can be used for either fluid or gas exchangeindependent of that in the bottom assembly (209). After the bottomassembly (209) and the top assembly (211) may be assembledindependently, cartilage grafts can be loaded into a culture well (162)with of without a confining ring (204). Then the top assembly may bescrewed down onto the bottom assembly to engage the o-ring (203) therebyeliminating leakage from the entire chamber as illustrated in FIG.23-FIG. 30.

FIG. 23 illustrates the application of mechanical stimulation byinducing compressive air/gas towards two flexible membranes (172 and193) that induce pressure on a cartilage graft sandwiched between twoporous platens (216 and 217) in a bioreactor filled with culture media.The cartilage grafts may be, but are not limited to, cartilage slices asillustrated in FIG. 23, or cartilage discs, or osteochondral plugs. Thecompression can be unconfined or confined. For confined compression asillustrated in FIG. 23, the cartilage slices can be seeded with cellsfirst, stacked together, and sandwiched between two porous platens (216and 217) in the confining ring (204). Recellularizable cells isolatedfrom autologous or allogenous sources may be seeded on the devitalizedcartilage grafts. Optionally, a centrifugal force or a positive pressurecan be applied to facilitate cell adhesion onto the devitalizedcartilage graft. The devitalized cartilage grafts can be recellularizedwith one or more than one type of cells from recellularizable cells. Theporous platens can be flat or with a curvature that can create a contouron the cartilage graft to match the contour of the defect site to berepaired in the recipient. The bottom porous platen (217) can have thesame curvature as the top porous platen (216) or can be flat. The porousplaten can be made of a group of materials such as titanium, stainlesssteel, biocompatible polymers, ceramics, hydroxyapatite, calciumphosphate, calcium sulfate, cancellous bone, or cortical bone.

The compressed air/gas can be driven by a piston and passes through port(188) through a Luer lock tubing connection (214) to compress theflexible membrane (193). Meanwhile, the compressed air/gas can also passthrough port (179) through a Luer lock tubing connection (214) tocompress the flexible membrane (172). The piston can be driven by acomputer controlled cam and/or stepper motor to move up and down tocreate a cyclic compression within the bioreactor. The pressure can bemonitor using two pressure gauges (110) and regulated by two valves(218). The compressed air/gas may be made of sterile 5% CO₂ in air. Thebioreactor may be able to fit into an incubator connected to one or twomedia reservoirs through ports (198, 199, 180, or 179). The cycliccompression can be carried out at pressure preferably of about 0 toabout 20 MPa, more preferably of about 0 and about 10 MPa, mostpreferably of about 0 and about 6 MPa, at a frequency preferably of fromabout 0.001 to about 5 Hz, more preferably of from about 0.1 to about 3Hz, and most preferably of from about 0.1 to about 1 Hz, for a period oftime preferably of from about 5 minutes to about 16 hours, morepreferably of from 5 minutes to about 8 hours, most preferably of fromabout 5 minutes to about 4 hours every day, and for a total durationpreferably of 1 to about 40 days, more preferably of 1 to about 28 days,most preferably of 1 to about 14 days. Alternatively, the cycliccompression can be conducted by inducing compression on the culturemedia directly to induce pressure on a cartilage graft sandwichedbetween two porous platens (216 and 217) with or without a confiningring (204) in a bioreactor filled with culture media as illustrated inFIG. 24. At the end of cultivation, the viable coherent stack ofcartilage slices or cartilage disc can be implanted along with orwithout the bottom porous platen (217).

FIG. 25 illustrates the application of the mechanical stimulation byinducing compressive stress using a load shaft (224) on a cartilagegraft sandwiched between two porous platens. The compression can beconfined or unconfined compression. The compression can also be carriedout under compressive stress control or displacement control. A spring(225) can be serially connected to the bottom of the load shaft (224) toallow larger range and better control of the movement of the loadingshaft during loading. The end of the spring can be flat and fixed ontothe top porous platen (226). The cartilage slices or discs can be seededwith recellularizable cells, stacked together, and sandwiched betweentwo porous platens (226 and 227) in the confining ring (204).Recellularizable cells isolated from autologous or allogenous sourcescan be seeded on the devitalized cartilage grafts before the applicationof mechanical stimuli. Optionally, a centrifugal force or a positivepressure can be applied to facilitate cell adhesion onto the devitalizedcartilage graft. The devitalized cartilage grafts can be recellularizedwith one or more than one type of cells from recellularizable cells. Theporous platens can be flat or with curvature that can create a contouron the cartilage graft to match the contour of the defect site to berepaired in the recipient. The bottom porous platen (227) can have thesame curvature as the top porous platen (226) or can be flat. The porousplaten can be made of a group of materials such as titanium, stainlesssteel, biocompatible polymers, ceramics, hydroxyapatite, calciumphosphate, calcium sulfate, cancellous bone, or cortical bone. Theloading shaft can be driven by a computer controlled cam and/or steppermotor to move up and down to create a cyclic compression within thebioreactor. The compressive stress can be monitored with a load cell(222) and the strain of the loading may be adjusted to obtain the targetstress. A flexible bellow (223) can be assembled between the top of theloading shafts (224) and the top chamber assembly (211) to preventcontamination during movements. The bioreactor may be able to fit intoan incubator and connected to one or two media reservoirs through ports(198, 199, 180, or 179). Gas exchange can be obtained through port(188), a Luer lock tube connecter (214), and a syringe filter (280).Under compressive stress control, the cyclic compression can be carriedout at compressive stress preferably of from about 0 to about 20 MPa,more preferably of from about 0 to about 10 MPa, most preferably of from0 to about 6 MPa, at a frequency preferably of from about 0.001 to about5 Hz, more preferably of from about 0.1 to about 3 Hz, and mostpreferably of from about 0.1 to about 1 Hz, for a period of timepreferably of from about 5 minutes to about 16 hours, more preferably offrom 5 minutes to about 8 hours, most preferably of from about 5 minutesto about 4 hours every day, and for a total duration preferably of 1 toabout 40 days, more preferably of 1 to about 28 days, most preferably of1 to about 14 days. Under displacement control, a dynamic displacementmay be superimposed on a static displacement. The static displacementcan be preferably from about 0 to about 20%, more preferably from about0 to about 10%, most preferably from about 0 to about 5% of thecartilage graft thickness. The cyclic compression can be carried out atdynamic displacement amplitude preferably of from about 0 to about 50%,more preferably of from about 0 to about 20%, most preferably of fromabout 0 to about 5% of the cartilage graft thickness, at a frequencypreferably of from about 0.001 to about 5 Hz, more preferably of fromabout 0.1 to about 3 Hz, and most preferably of from about 0.1 to about1 Hz, for a period of time preferably of from about 5 minutes to about16 hours, more preferably of from 5 minutes to about 8 hours, mostpreferably of from about 5 minutes to about 4 hours every day, and for atotal duration preferably of 1 to about 40 days, more preferably of 1 toabout 28 days, most preferably of 1 to about 14 days. At the end ofcultivation, the coherent stack of cartilage slices or cartilage discmay be implanted along with or without the bottom porous platen (227).

Before applying mechanical stimuli, cell seeding on osteochondral plugscan be conducted outside of a bioreactor. Alternatively, cell seedingcan be conducted directly in the bioreactor as illustrated in FIG. 26and FIG. 27.

The cartilage cap and the bone portion of the devitalized osteochondralplug can be recellularized with the same type of cells. Alternatively,the cartilage cap and the bone portion of the devitalized osteochondralplug can be recellularized with different type of cells.Recellularizable cells isolated from autologous or allogenous sourcescan be seeded on the devitalized cartilage grafts before application ofmechanical stimuli. Optionally, a centrifugal force or a positivepressure can be applied to facilitate cell adhesion onto the devitalizedcartilage graft. The cartilage cap of the devitalized osteochondral plugcan be recellularized with one or more than one type of cells fromrecellularizable cells. The bone portion of the devitalizedosteochondral plug can be recellularized with one or more than one typeof cells from recellularizable cells.

As illustrated in FIG. 26, when any one of the osteochondral plugs inFIG. 2-FIG. 5 is fit into the culture well (162), the inferior surfacefacing the osteochondral bone portion of the cartilage cap (6, 37, 39,41, 43 or 45) can be placed against the top surface of a porous hollowcylinder (278). The bone portion can be fit into the middle hole (166)of the culture well (162) with the rubber o-ring (234) on the peripheralsurface that creates a seal. Cell suspension can be either directlyinjected or driven by a pump through a port (230), and through a rigidfeeding tube (232) and the sprinkle head (233) to spray the cells ontothe cartilage cap of the osteochondral plug. The bioreactor can also beturned upside down with the osteochondral plug secured in the culturewell and the bone portion facing up. In this configuration, cellsuspension that can be the same or different from the cell suspensionfor the cartilage cap recellularization may be either directly injectedor driven by a pump through a port (230), and through a rigid feedingtube (237) and the sprinkle head (233) to spray the cells onto the boneportion of the osteochondral plug.

In another embodiment, the bioreactor can be placed horizontally asillustrated in FIG. 27 with an osteochondral plug secured in the culturewell as described above. Cell suspension for cartilage caprecellularization can be injected or driven by a pump through a port(198) and a rigid feeding tube (240) onto the circumferential surface ofthe cartilage cap. Cell suspension for the bone portionrecellularization can be injected or driven by the pump through a port(171) and a rigid feeding tube (239) onto the circumferential surface ofthe bone portion. The cell seeding system as illustrated FIG. 26 andFIG. 27 can be applied in conjunction with the bioreactor systems toreplenish fresh cells between or during compression regime.

After cell seeding, the osteochondral plug can be cultured undercompression with a loading shaft with or without a spring seriallyattaching to as illustrated in FIG. 28. The bottom of the osteochondralplug can be supported by a supporting ring (248) that may be screwedinto the bottom of the culture well (162) during compression. Thecartilage cap of the osteochondral plug can be placed between a porousplaten (226) and a porous ring (241) in a confining ring (204). Theporous platen or the porous ring can be made of a group of materialssuch as titanium, stainless steel, biocompatible polymers, ceramics,hydroxyapatite, calcium phosphate, calcium sulfate, cancellous bone, orcortical bone. A spring (225) can be serially connected to the bottom ofthe load shaft (224) to allow larger range and better control of themovement of the loading shaft during loading. The end of the spring canbe flat and fixed onto the top porous platen (226). Alternatively, theloading shaft can directly compress on the cartilage cap using a solidbead (243) and a porous platen (226) to ensure the centerline of theloading shaft being parallel to the centerline of the osteochondral plugto be compressed as illustrated in the right panel of FIG. 28. Theloading shaft can be driven by a computer controlled cam and/or steppermotor to move up and down to create a cyclic compression within thebioreactor. The compressive stress can be monitored with a load cell(222) and the strain of the loading can be adjusted to obtain the targetstress. A flexible bellow (223) can be assembled between the top of theloading shafts (224) and the top chamber assembly (211) to preventcontamination during movements. The bioreactor may be able to fit intoan incubator and connected to a media reservoir through ports (198, 199,180, 179). Gas exchange can be obtained through port (188), a Luer locktube connecter (214), and a syringe filter (280). Under compressivestress control, the cyclic compression can be carried out at compressivestress preferably of from about 0 to about 20 MPa, more preferably offrom about 0 to about 10 MPa, most preferably of from 0 to about 6 MPa,at a frequency preferably of from about 0.001 to about 5 Hz, morepreferably of from about 0.1 to about 3 Hz, and most preferably of fromabout 0.1 to about 1 Hz, for a period of time preferably of from about 5minutes to about 16 hours, more preferably of from 5 minutes to about 8hours, most preferably of from about 5 minutes to about 4 hours everyday, and for a total duration preferably of 1 to about 40 days, morepreferably of 1 to about 28 days, most preferably of 1 to about 14 days.

If desired, osteochondral plugs seeded with cells can be compressed withcartilage caps opposite each other as illustrated in FIG. 29 and FIG.30. The compression can be either confined or unconfined. The bottom ofthe first osteochondral plug can be supported by a supporting ring (248)that may be screwed into the bottom of the culture well (162) duringcompression. The second osteochondral plug can be placed on top of thefirst osteochondral plug and the superficial surface of the cartilagecap of the osteochondral plugs may be placed opposing each other. Forconfined compression, cartilage caps from both osteochondral plugs canbe placed in a confining ring (247) with or without a porous platen(226) in between (FIG. 29). If congruent contoured surfaces between twoosteochondral plugs are desired, a porous platen (279) that has thetarget curvature according to the contour of the recipient joint (convexor concave surfaces) as illustrated in the left panel of FIG. 30 can beused between the two opposing osteochondral plugs.

Alternatively, a mold that has a desired curvature can be used toreplace one of the osteochondral plugs as illustrated in the right panelof FIG. 30. The mold can be made of a porous material that may be madeof a group of materials such as titanium, stainless steel, biocompatiblepolymers, ceramics, hydroxyapatite, calcium phosphate, calcium sulfate,cancellous bone, or cortical bone. The loading shaft can directlycompress on the bone portion of the second osteochondral plug or a moldthrough a solid bead (243) and a porous platen (226) to ensure thecenter line of the loading shaft being parallel to the centerline of theosteochondral plugs to be compressed.

The loading shaft can be driven by a computer controlled cam and/orstepper motor to move up and down to create a cyclic compression withinthe bioreactor. The compressive stress can be monitored with a load cell(222) and the strain of the loading can be adjusted to obtain the targetstress. A flexible bellow (223) can be assembled between the top of theloading shafts (224) and the top chamber assembly (211) to preventcontamination during movements. The bioreactor may be able to fit intoan incubator and connected to a media reservoir through ports (198, 199,180, or 179). Gas exchange can be obtained through port (188), a Luerlock tube connecter (214), and a syringe filter (280). Under compressivestress control, the cyclic compression can be carried out at compressivestress preferably of from about 0 to about 20 MPa, more preferably offrom about 0 to about 10 MPa, most preferably of from 0 to about 6 MPa,at a frequency preferably of from about 0.001 to about 5 Hz, morepreferably of from about 0.1 to about 3 Hz, and most preferably of fromabout 0.1 to about 1 Hz, for a period of time preferably of from about 5minutes to about 16 hours, more preferably of from 5 minutes to about 8hours, most preferably of from about 5 minutes to about 4 hours everyday, and for a total duration preferably of 1 to about 40 days, morepreferably of 1 to about 28 days, most preferably of 1 to about 14 days.

In another embodiment of the current invention, cartilage discs or stackof slices can be recellularized and cultured in vitro in a bioreactor asdescribed. Meanwhile, the bone plug that may be cleaned and disinfectedwithout cartilage tissue attached, and/or bony material made from, forexample, demineralized bone matrix, hydroxyapatite, ceramics, calciumphosphate, or calcium sulfate in the form of cylinders can berecellularized and cultured separately from the cartilage discs orslices in a bioreactor. After culturing in separation for certainduration, the soft tissue, i.e., the cartilage discs or slices, and thehard tissue, i.e., the bony tissue can be assembled together to beimplanted directly or further cultured in a bioreactor to form acomposite osteochondral cartilage grafts.

If desired, the devitalized osteochondral plugs, cartilage discs, orcartilage slices can be recellularized in vivo. In one embodiment, thedevitalized cartilage grafts can be implanted in a recipient's own softtissue, for example, under a muscle pouch or a fat pad or other softtissue containing progenitor or stromal cells for about 7 days to about3 months. Optionally, before the soft tissue implantation, thedevitalized cartilage grafts can be seeded with cells from one or morethan one type of cells from recellularizable cells. The devitalizedcartilage graft can also be treated with chemical stimuli before orafter the in vivo soft tissue implantation. In addition, centrifugalforce or positive pressure can be optionally applied to facilitate celladhesion onto the devitalized cartilage graft. Before implanting intothe cartilage defect site in the recipient, the implanted cartilagegrafts may be retrieved from the soft tissue, trimmed off the excessivefibrous tissue if present surrounding the recellularized cartilagegraft, and rinsed with an isotonic solution, such as isotonic saline.Then the in vivo recellularized graft can be implanted into the targetdefect site.

Before implantation, a first bore at the cartilage defect site may becreated down into the osteochondral bone to remove the damaged cartilagetissue and underlying bone in the recipient. In one aspect, the diameterof the first bore matches the maximum diameter of the bone portion ofthe osteochondral plug if the osteochondral plug may be chosen to beused as a graft. The length of the first bore can be the same as theosteochondral plug to be implanted. Then, a second shaped bore, such asa star-shaped bore, may be created at the cartilage portion of the firstbore. The second shaped bore may be concentric to and on top of thefirst bore. The second shaped bore can be crafted using a customdesigned coring device as illustrated in FIG. 31a . The coring devicemay be composed of a star-shaped cutter (259) to match the shape andsize of the cartilage cap of an osteochondral plug, cartilage disc, orcartilage slices to be implanted, and an adaptor (260) to assist thecoring.

After the custom designed coring device cuts through the cartilagetissue and reaches the bone, the adaptor (260) can be removed with thehelp of a pushing device (67 in FIG. 31a ) and the star-shaped cutter(259) remains in place FIG. 31b . The star-shaped cutter can be used asa boundary for removing the damaged cartilage tissue within thestar-shaped cutter from the recipient to create a star-shaped bore inthe cartilage portion of the recipient defect site. The star-shapedcutter may be designed so that its outer surface may be straight andmatches the size and shape of the cartilage portion of the cartilagegraft to be implanted (FIG. 32). The bottom portion of the inner surfaceof the star-shaped cutter may be angled to form a beveled sharp cuttingedge (261).

In one embodiment, an osteochondral plug (with or withoutrecellularization in situ, in vitro, or in vivo) can be used to repairthe defect site as illustrated in FIG. 34. The osteochondral plug may beselected to match the size, contour, and location of the defect site. Abonding agent, such as a photoactive dye, can be dissolved in anisotonic solution, such as isotonic saline or phosphate buffered saline.The shaped second bore on the cartilage tissue along with the first boreat the bone portion of the recipient can be filled with the photoactivedye for 5-10 minutes. Then the photoactive dye may be removed and thefirst bore in the bone portion is optionally rinsed with an isotonicsolution, such as isotonic saline.

If a step cylindrical osteochondral plug is used as a graft, theosteochondral plug can fit tightly into the first bore and supported bythe wall of the bone portion of the first bore. Alternatively, if thediameter of the bone portion of the step cylindrical osteochondral plugis slightly smaller than the diameter of the first bore in the boneportion of the recipient, a bone filler can be inserted into the boneportion of the first bore that is created at the defect site to fill thegap between the wall of the first bore and the bone portion of theosteochondral plug. The bone filler can also be inserted into the firstbore to create a flat surface at the bottom of the first bore so that itcan provide support for the osteochondral plug. Meanwhile, the same bonefiller can be inserted into the gaps or channels or slots (if present)on the osteochondral plug. In addition, if the cartilage cap of the stepcylindrical osteochondral plug fits loosely into the second bore, acartilage filler can be applied in the gap between the peripheral of thecartilage cap of the osteochondral plug and the second shaped bore. Thecartilage filler can also be inserted into gaps or a bore or channels orslots on the cartilage cap from the bottom of the osteochondral plug ifsuch gaps or bore or channels or slots are present. The same or adifferent photoactive dye used to treat the bores created at therecipient cartilage defect can be used to treat the circumferential areaof the cartilage cap of the osteochondral plug. The superficial surfaceof the osteochondral plug can be at the same height as the surface ofthe surrounding recipient cartilage surface. If desired theosteochondral plugs can be applied in combination with the cartilagediscs or slices or flakes to match the depth and/or contour of therecipient cartilage and to optimize the repair process.

In another embodiment, a cartilage disc (with or withoutrecellularization in situ, in vitro, or in vivo) can be used to repairthe defect site as illustrated in FIG. 34. The cartilage disc may beselected to match the size, contour, and location of the defect site. Abonding agent, such as a photoactive dye, can be dissolved in anisotonic solution, such as isotonic saline. The shaped second bore onthe cartilage tissue along with the first bore at bone portion of therecipient can be filled with the photoactive dye for 5-10 minutes. Thenthe photoactive dye may be removed and the first bore in the boneportion is optionally rinsed with an isotonic solution, such as isotonicsaline. A bone filler can be used to fill up the bone portion of thefirst bore to provide support for the cartilage disc. The cartilage disccan fit tightly into the shaped second bore. Alternatively, if thecartilage disc fits loosely into the second bore, a cartilage filler canbe applied in the gap between the peripheral of the cartilage disc andthe second shaped bore. The cartilage filler can also be inserted into abore or channels or slots on the cartilage disc from the bottom of thecartilage disc if such bore or channels or slots present. The same or adifferent photoactive dye used to treat the bores created at therecipient cartilage defect can be used to treat the circumferential areaof the cartilage disc. The cartilage disc can be inserted into thedefect site with the cartilage disc being at the same height as thesurrounding recipient cartilage. If desired the cartilage discs can beapplied in combination with the osteochondral plugs, cartilage slices orflakes to match the depth and/or contour of the recipient cartilage andto optimize the repair process.

In yet another embodiment, cartilage slices (with or withoutrecellularization in situ, in vitro, or in vivo) can be used to repairthe defect site. The cartilage slices can be tailored according to thesize, contour, and location of the bore created at the cartilage defectsite. A bonding agent, such as a photoactive dye, can be dissolved in anisotonic solution, such as isotonic saline. The shaped second bore onthe cartilage tissue along with the first bore at bone portion of therecipient can be filled with the photoactive dye for 5-10 minutes. Thenthe photoactive dye may be removed and the first bore in the boneportion is optionally rinsed with an isotonic solution, such as isotonicsaline. A bone filler can be used to fill up the bone portion of thefirst bore to provide support for the cartilage slices. The cartilageslices may fit tightly into the shaped second bore. Alternatively, ifthe cartilage slices fit loosely into the second bore, a cartilagefiller can be applied in the gap between the peripheral of the cartilageslices and the second shaped bore. The same or a different photoactivedye used to treat the bores created at the recipient cartilage defectcan be used to treat the circumferential area of the cartilage slices.The shaped cartilage slices can be stacked together, optionally a secondbonding agent and/or with viable cells seeded between the slices, andinserted into the defect site until at the same height as thesurrounding cartilage. The second bonding agent may be the same ordifferent from the bonding agent used to treat the circumferential areaof the cartilage slices. If desired the cartilage slices can be appliedin combination with the osteochondral plugs, cartilage discs or flakesto match the depth and/or contour of the recipient cartilage and tooptimize the repair process.

In another embodiment, cartilage curls or flakes (with or withoutrecellularization in situ, in vitro, or in vivo) can be used to repairthe cartilage defect site. The cartilage curls or flakes can also beapplied in combination with cartilage slices, discs or osteochondralplugs to repair the cartilage defect site. The cartilage curls or flakesmay be applied directly or mixed with a matrix, such as demineralizedbone matrix, and/or a carrier, such as hyaluronic acid, isotonic saline,phosphate buffered saline or bone marrow from the implant recipient toform cartilage filler. A bone filler can be used to fill up the boneportion of the first bore to provide support for the cartilage flakes orcurls. Then the cartilage curl or flake filler, in a form such as aputty or gel, can be placed into the cartilage defect site directly orinjected into the cartilage defect site through a syringe that may beconnected to a stent or needle. A stack of cartilage slices or acartilage disc can be placed on top of the cartilage flake or curlfiller with the superficial surface of the stack of the cartilage sliceor the cartilage disc being at the same height as the surface of thesurrounding recipient cartilage.

The bone filler can be a mixture of a matrix with or without a carrier.The bone filler can be in the format of a sheet, a disc, a tape, asponge, a cube, a solid or hollow cylinder, particles, gel, or putty.The matrix may be one or more of, for example, autologous crushed boneharvested from the defect site; demineralized bone matrix; cancellousand cortical bone mixture; small intestine submucosa, amniotic membrane,ligament, tendon, skin, muscle tissue, periostieum, or synovial tissue;ceramics; hydroxyapatite; calcium phosphate; calcium sulfate; poroussurgical grade titanium or stainless steel; or any combination of theabove. The carrier may be one or more of, for example,dihydroxyphenylalanine (DOPA) based adhesive, glucose, concentratedalbumin, cyanoacrylate adhesive, gelatin-resorcin-formalin adhesive,chondroitin sulfate aldehyde N-acetylglucosamine (GlcNAc), mussel-basedadhesive, poly(amino acid)-based adhesive, cellulose-based adhesive,synthetic acrylate-based adhesives, platelet rich plasma (PRP),monostearoyl glycerol co-Succinate (MGSA), monostearoyl glycerolco-succinate/polyethylene glycol (MGSAPEG) copolymers, or a combinationcomprising at least one of the foregoing polymers. The carrier can alsobe one or more of, for example, native or modified collagen, gelatin,agarose, modified hyaluronic acid, fibrin, chitin, biotin, avidin,native or crosslinked chitosan, alginate, demineralized bone matrix,MATRIGEL®, HUMAN EXTRACELLULAR MATRIX®, homogenized connective tissue,proteoglycans, fibronectin, laminin, fibronectin, elastin, heparin,glycerol, or a combination comprising at least one of the foregoingpolymers. The carrier may include bioactive growth supplements such asFGF-family, TGF-family, IGF-1, PDGF, EGF, VEGF, HGF, PTHrP, Ihh,dexamethasone, insulin, transferrin, selenium, ITS, or ascorbate. Thecarrier may also include bioactive growth supplements from theextractions of demineralized bone matrix, basement membrane, orsubmucosa matrix. The carrier may include cytokines, for example, anILαR antibody, TNF-a receptor antagonist, cyclooxygenase-2 specificinhibitors, MAP kinase inhibitors, NO synthase inhibitors, NF-κBinhibitors, or inhibitors of MMP. Moreover, the carrier may also includeone or more than one type of cells from recellularizable cells. The bonefiller may also be a cortical and/or cancellous bone plug.

The cartilage filler may be a mixture of a matrix with or without acarrier. The cartilage filler can be in the format of a sheet, a disc, atape, a sponge, a cube, a solid or hollow cylinder, particles, gel, orputty. The matrix in the cartilage filler may be one or more of, forexample, demineralized bone matrix; small intestine submucosa, amnioticmembrane, ligament, tendon, skin, muscle tissue, periostieum, synovialtissue, or devitalized cartilage curls and flakes; or any combination ofthe above. The carrier in the cartilage filler may be one or more of,for example, dihydroxyphenylalanine (DOPA) based adhesive, glucose,concentrated albumin, cyanoacrylate adhesive, gelatin-resorcin-formalinadhesive, chondroitin sulfate aldehyde N-acetylglucosamine (GlcNAc),mussel-based adhesive, poly(amino acid)-based adhesive, cellulose-basedadhesive, synthetic acrylate-based adhesives, platelet rich plasma(PRP), monostearoyl glycerol co-Succinate (MGSA), monostearoyl glycerolco-succinate/polyethylene glycol (MGSAPEG) copolymers, or a combinationcomprising at least one of the foregoing polymers. The carrier in thecartilage filler may be one or more of, for example, native or modifiedcollagen, gelatin, agarose, modified hyaluronic acid, fibrin, chitin,biotin, avidin, native or crosslinked chitosan, alginate, demineralizedbone matrix, MATRIGEL®, HUMAN EXTRACELLULAR MATRIX®, homogenizedconnective tissue, proteoglycans, fibronectin, laminin, fibronectin,elastin, heparin, glycerol, or a combination comprising at least one ofthe foregoing polymers. The carrier in the cartilage filler may be oneor more of, for example, polymethylmethacrylate, polyurethane,acryloilmorpholine, N,N-dimethyl acrylamide, N-vinyl pyrrolidone andtetrahydrofurfuryl methacrylate, hydroxyapatite, cross-linkage orfunctionalization of hyaluronan-based collagen and alginate,polyurethane, or polylactic acid. The carrier in the cartilage fillermay include one or more of, for example, FGF-family, TGF-family, IGF-1,PDGF, EGF, VEGF, HGF, PTHrP, Ihh, dexamethasone, insulin, transferrin,selenium, ITS, or ascorbate. The carrier in the cartilage filler mayinclude one or more of, for example, bioactive growth supplementsextracted from demineralized bone matrix, basement membrane, orsubmucosa matrix. The carrier in the cartilage filler may include one ormore photoactive agents, for example, a xanthene dye, naphthalimidecompounds, riboflavin-5-phosphate, N-hydroxypyridine-2-(1H)-thione,N-(20-ethylaminoethyl)-4-amino-1,8-naphthalimide,bis-diazopyruvamide-N,N9-bis(3-diazopyruvoyl)-2,29-(ethylenedioxy)bis-(et-hylamine)(DPD), diazopyruvoyl (DAP), methylene blue, erythrosin, phloxime,thionine, methylene green, rose Bengal, acridine orange, xanthine dye,thioxanthine dyes, ethyl eosin, eosin Y, or a combination comprising atleast one of the foregoing photoactive groups. The carrier in thecartilage filler may include one or more antioxidants, for example,sodium nitroprusside, cartilage matrix glycoprotein (CMGP), vitamins C,vitamin E, selenium, N-Acetylcysteine (NAC) estradiol, glutathione,melatonin, resveratrol, flavonoid, carotene, aminoguanidine, orlycopene. The carrier in the cartilage filler may include one or morecrosslinking agents, for example, glutaraldehyde; glyceraldehyde;genipin; glucose or ribose; poly(ethylene glycol) diepoxide crosslinker;poly(ethylene glycol) diglycidyl ether; EDC and NHS; transglutaminase;ethylenediamine; lysyl oxidase family; hexamethylene diisocyanate(HMDIC); dimethyl suberimidate (DMS);dimethyl-3-3′-dithiobispropionimidate (DTBP), or acryl azide. Thecarrier may include cytokines, for example, an IL-1αR antibody, TNF-areceptor antagonist, cyclooxygenase-2 specific inhibitors, MAP kinaseinhibitors, NO synthase inhibitors, NF-κB inhibitors, or inhibitors ofMMP. The carrier in the cartilage filler may also include one or morethan one type of cells from recellularizable cells.

The bonding agent can be one or more of photoactive dye(s) which can be,but are not limited to, xanthene dye, naphthalimide compounds,riboflavin-5-phosphate, N-hydroxypyridine-2-(1H)-thione,N-(2′-ethylaminoethyl)-4-amino-1,8-naphthalimide,bis-diazopyruvamide-N,N9-bis(3-diazopyruvoyl)-2,29-(ethylenedioxy)bis-(et-hylamine)(DPD), diazopyruvoyl (DAP), methylene blue, erythrosin, phloxime,thionine, methylene green, rose Bengal, acridine orange, xanthine dye,thioxanthine dyes, ethyl eosin, eosin Y, and a combination comprising atleast one of the foregoing photoactive groups.

The bonding agent may include one or more of, for example,hyaluronidase, chondroitinase, collagenase, trypsin, superoxidedismutase (SOD), or catalase. The bonding agent may include one or moreof bioactive growth supplements from the extractions of demineralizedbone matrix, basement membrane, or submucosa matrix. The bonding agentmay include one or more of bioactive growth supplements such asFGF-family, TGF-family, IGF-1, PDGF, EGF, VEGF, HGF, PTHrP, Ihh,dexamethasone, insulin, transferrin, selenium, ITS, or ascorbate. Thebonding agent may include one or more of, for example,dihydroxyphenylalanine (DOPA) based adhesive, glucose, concentratedalbumin, cyanoacrylate adhesive, gelatin-resorcin-formalin adhesive,chondroitin sulfate aldehyde N-acetylglucosamine (GlcNAc), mussel-basedadhesive, poly(amino acid)-based adhesive, cellulose-based adhesive,synthetic acrylate-based adhesives, platelet rich plasma (PRP),monostearoyl glycerol co-Succinate (MGSA), monostearoyl glycerolco-succinate/polyethylene glycol (MGSAPEG) copolymers, or a combinationcomprising at least one of the foregoing polymers. The bonding agent mayinclude one or more of, for example, collagen, gelatin, agarose,modified hyaluronic acid, fibrin, chitin, biotin, avidin, native orcrosslinked chitosan, alginate, demineralized bone matrix, MATRIGEL®,HUMAN EXTRACELLULAR MATRIX®, homogenized connective tissue,proteoglycans, fibronectin, laminin, fibronectin, elastin, heparin,glycerol, or a combination comprising at least one of the foregoingpolymers. The bonding agent may include one or more of, for example,polymethylmethacrylate, polyurethane, acryloilmorpholine, N,N-dimethylacrylamide, N-vinyl pyrrolidone and tetrahydrofurfuryl methacrylate,hydroxyapatite, cross-linkage or functionalization of hyaluronan-basedcollagen and alginate, polyurethane, or polylactic acid. The bondingagent may include one or more of, for example, sodium nitroprusside,cartilage matrix glycoprotein (CMGP), vitamins C, vitamin E, selenium,N-Acetylcysteine (NAC) estradiol, glutathione, melatonin, resveratrol,flavonoid, carotene, aminoguanidine, or lycopene. The bonding agent mayinclude one or more of, for example, glutaraldehyde; glyceraldehydes;genipin; glucose or ribose; poly(ethylene glycol) diepoxide crosslinker;poly(ethylene glycol) diglycidyl ether; EDC and NHS; transglutaminase;lysyl oxidase family; hexamethylene diisocyanate (HMDIC); dimethylsuberimidate (DMS); dimethyl-3-3′-dithiobispropionimidate (DTBP); oracryl azide. The bonding agent may also include one or more than onetype of cells from recellularizable cells.

The cartilage graft such as osteochondral plugs, cartilage discs,cartilage slices, or cartilage flakes or curls as described above can becleaned, disinfected, and devitalized; or cleaned, disinfected,devitalized, and recellularized in situ, in vitro, or in vivo.

In one embodiment, in order to easily insert the cartilage graft (suchas osteochondral plug, or cartilage disc, or cartilage slices) into thebore created at the recipient cartilage defect site and minimize thecompressive force applied on the cartilage graft during insertion, aninsertion device (253) can be applied (FIG. 33). A thin needle (254)attached to a syringe of the insertion device (253) can penetrate thecartilage portion of a cartilage graft and transfer the cartilage graftinto the bore that may be created on the defect site until thecircumferential surface of the cartilage cap on the osteochondral plugor cartilage disc or a stack of cartilage slices becomes interferencewith the recipient tissue. Then the needle may be inserted furtherthrough the cartilage portion until it reaches the underlying bone ofthe recipient tissue or bone filler if present.

A vacuum device (257) or a plunger (258) can be applied to remove theair/gas and/or fluid trapped inside of the bore to allow ambientpressure above the graft to push the cartilage graft into said defectsite. The bore created in the recipient tissue at the defect site can bea straight (155) or step cylindrical (255) shape as illustrated in FIG.33. If a step cylindrical shape osteochondral plug may be used as graftand fit into a straight bore, a bone filler can fill in the gap betweenthe bone portion of the step cylindrical osteochondral plug and the wallof the bore (155) in the bone portion.

Alternatively, if a step cylindrical shape osteochondral plug may beused as a graft and fit into a step cylindrical bore (255), theosteochondral plug may be tightly fit into the bore. If gaps, or a bore,or channels, or slots are crafted on the bone portion of theosteochondral plug, the gaps or a bore or channels or slots can befilled with the same bone filler as described above. In all cases, thecartilage cap of an osteochondral plug, or cartilage disc, or cartilageslices can be tightly fit into the bore and supported by the wall ofeither the bone or cartilage portion of the bore. The superficialsurface of the osteochondral plug, cartilage disc, or cartilage slicesmay be at the same height as the surface of the surrounding recipientcartilage surface. If desired, bone filler can also be injected into thebone portion of the bore in the recipient through the same needle on theinsertion device after the cartilage graft has been properly inserted.

After insertion of the cartilage grafts and a time period of about 2 toabout 10 minutes, the photoactivated dye, if chosen as one of thebonding agents, can be activated by a laser as illustrated in FIG. 34.The laser wave length can be from long ultraviolet 250 nm to farinfrared 900 nm depending on the type of photoactive dye that is used.The laser beam can be delivered through an optical fiber with a spotsize of about 500 micrometer to about 5 mm. The power fluence of thelaser is about 10 to about 800 J/cm² and the irradiation/exposure timecan be between about 30 sec to about 30 minutes. The interface betweenthe boundaries of the cartilage being repaired (266) and the cartilagegraft (36 or 37 as illustrated in FIG. 34) being used in the repair canbe sealed with the surrounding recipient cartilage tissue by thisphotoactivated integration. If viable cells are present in the cartilagegrafts, in order to prevent the phototoxicity of the photoactive dye,the cartilage matrix can be optionally soaked with antioxidants, such aslycopene, to protect viable cells presented in the in vitro, in vivo, orin situ recellularized devitalized cartilage graft. Alternatively, oneor more antioxidants can be included in the photoactive dye solution toprevent phototoxicity.

Since the delivery system of the laser beams can be small, the proceduredescribed above can be used for both open knee surgery (FIG. 34) andminimally invasive arthroscopic surgery, such as the procedure ofrepairing the cartilage defect on the femoral condyle illustrated inFIG. 35-FIG. 37. During an arthroscopic surgery, a shaped bore can becreated using a shaped coring device (259 and 268), a photoactive dyecan be applied in the shaped bore and on the circumferential surface ofthe cartilage graft, cartilage graft can be inserted into the shapedbore using a insertion device, and an energy source can be applied toactivate the photoactive dye to seal the interface between the interfaceof the recipient cartilage being repaired and the cartilage graft.

Optionally, in addition to sealing the interface between the recipientcartilages being repaired the cartilage graft with photoactivatedcrosslinking, the bore created on the defect site of the recipientcartilage tissue and the cartilage graft can be coated with additionalbonding agents, such as crosslinking agents. Crosslinking agents can beused to facilitate integration of the cartilage graft and thesurrounding tissue after implantation and to restore the normal fluiddynamics environment of the cartilage tissue. The crosslinking agentscan be chemical or enzymatic and can be, but are not limited to,glutaraldehyde; glyceraldehyde; genipin; glucose or ribose;poly(ethylene glycol) diepoxide crosslinker; poly(ethylene glycol)diglycidyl ether; EDC and NHS, transglutaminase; lysyl oxidase family;hexamethylene diisocyanate (HMDIC); dimethyl suberimidate (DMS);dimethyl-3-3′-dithiobispropionimidate (DTBP), or acryl azide.

EXAMPLES Example 1 Osteochondral Plug, Straight, Step, or Dumbbell Shape

The distal end of a human femur was procured from a suitable donor,transported on wet ice to the processing facility. A picture was takenand was superimposed on a customer made grid/coordinate system to createa map of the human femoral condyle. The femoral condyle end was “cored”with a coring device or drilled with a hollow cylindrical drill bit toproduce multiple cylindrical osteochondral plugs with diameter rangefrom 5-20 mm and the length of the bone portion from 5-20 mm. Thecoordinate of each individual cylindrical plug was recorded according tothe map. The cylindrical plugs were rinsed with isotonic saline. Thenone of the cylindrical plugs was inserted into a holder, such asillustrated in FIG. 7, with the cartilage cap positioned face down andsupported by the custom made bolt (60) as illustrated in FIGS. 7(d, eand f). The length of the bone portion of the osteochondral plugprotruding above the top of the holder was adjusted by the custom madebolt. Then set screws (57), preferably to be oriented 90 degrees apart,were engaged to further secure the osteochondral plug within the holder(63) and to adjust the centerline of the osteochondral plug to beparallel to the cutting tool centerline or cutting direction. The holderwas fit into the headstock on a lathe. The end of the bone portion wastrimmed so that the bottom surface of the bone portion was parallel tothe superficial surface of the cartilage cap.

For crafting a dumbbell shape osteochondral plug, 5 mm length of thebone portion right underneath of the cartilage cap of the straightosteochondral plug was cut on a lathe so that the diameter of cutportion was about 70% of the rest part of the osteochondral plug. Forcrafting a step cylindrical shape osteochondral plug, the entire boneportion of the straight osteochondral plug was cut on a lathe so thatthe diameter of the bone portion was smaller than that of the cartilagecap of the osteochondral plug. During crafting, isotonic saline wassprayed on the graft through a cooling system installed on the lathe.

Example 2

Osteochondral Plug with Gaps, Hollow Cylinder, or Multiple SmallCylindrical Channels

The osteochondral plugs, crafted to be straight, step cylindrical, ordumbbell shape as illustrated in Example 1 can be further crafted tohave channels, gaps, or slots, such as osteochondral plugs (8 a, 8 b,10, or 14; 22 a, 22 b, 23, or 25; 30 a, 30 b, 31, or 33) illustrated inFIG. 2-FIG. 4. Before being inserted into a holder (63 in FIG. 7), thelength of the bone portion of the osteochondral plug was measured. Then,an osteochondral plug, e.g. a dumbbell shape cylindrical plug with 14 mmmaximum diameter and 10 mm minimum diameter, was inserted into a holderwith the cartilage cap positioned to face down and supported by thecustom made bolt (60), as illustrated in FIG. 7(f).

The length of the bone portion of the osteochondral plug protrudingabove the top of the holder was adjusted by the custom made bolt (60).Then set screws (57), preferably to be oriented 90 degrees apart, wereengaged to further secure the osteochondral plug within the holder (63)and to adjust the centerline of the osteochondral plug to be parallel tothe cutting tool centerline or cutting direction. The holder was fitvertically, i.e., with the osteochondral bone portion facing up, intothe clamp fixed on the x-y table of the drilling/milling machine so thatit could move in a horizontal or cross direction.

An osteochondral plug with gaps as illustrated in 22 a in FIG. 3 wascrafted by adjusting the holder's height so that the cartilage and boneinterface of the osteochondral plug was at the same height of the end ofthe endmill. Alternatively, the end of the endmill may be adjusted to beat the same height as the position chosen at the deep, middle orsuperficial region of the cartilage cap of the osteochondral plug, ifthe gaps are designed to occupy portion of the cartilage cap. Thediameter of the endmill was 5 mm and smaller than the width of the slots(64) on the holder. By moving the holder (63) horizontally along the xdirection, the endmill moved through the slots created on the holder andcut through the bone portion of the osteochondral to obtain a gap (9).Again, by moving the holder along the y direction, the endmill movedthrough the slots (64) created on the holder and cut through the boneportion of the osteochondral to obtain another gap (9) so that two gapsform 90 degree angles along the entire length of the bone portion up tothe cartilage and osteochondral bone interface. Similar millingprocedures were conducted to craft gaps that were parallel to the centerline of the osteochondral plug and parallel to each other (22 b) asillustration in FIG. 3.

The osteochondral plug with a hollow cylinder on the bone portion (23)as illustration in FIG. 3 was crafted by adjusting the holder fixed on aclamp on an x-y table so that the centerline of the cylindrical boneportion of the osteochondral plug was the same as that of the drillingbit. The diameter of the drill bit was chosen to be 8 mm. The centerhole was first crafted by drilling down with a drill bit. The depth ofthe drill bit traveled was set to be the same as the length of the boneof portion of the osteochondral plug. After finishing drilling, the flatend of the center hole was created by milling with an endmill that hasthe same diameter as the drill bit. Alternatively, the end of theendmill may be adjusted to be at the same height as the position chosenat the deep, middle or superficial region of the cartilage cap of theosteochondral plug, if the center hole is designed to occupy portion ofthe cartilage cap.

The osteochondral plug with multiple small channels (15) along the wholelength of the bone portion up to the cartilage and osteochondral boneinterface (25) as illustrated in FIG. 3 was crafted by adjusting theholder fixed on a clamp on an x-y table so that the centerline of thecylindrical bone portion of the osteochondral plug was parallel to thedrilling bit. The diameter of the drill bit was chosen to be 1 mm. Thecenter of the first drilling was along the centerline of theosteochondral plug. The rest of the drilling centers were on the circleof 6 mm diameter from the first drilling center and 60 degree apartalong the circle. The depth of the drill bit traveled was set to be thesame as the length of the bone portion of the osteochondral plug. Afterfinishing drilling, the flat end of the channels was created by millingwith an endmill that has the same diameter as the drill bit.Alternatively, the end of the endmill may be adjusted to be at the sameheight as the position chosen at the deep, middle or superficial regionof the cartilage cap of the osteochondral plug, if the channels aredesigned to occupy portion of the cartilage cap. During crafting,isotonic saline was sprayed on the graft through a cooling systeminstalled on the milling/drilling machine.

Example 3

Osteochondral Plug with Channels at the Cartilage/Bone Interface

The osteochondral plugs, crafted to be straight, step cylindrical, ordumbbell shape as illustrated in Example 1 can be further crafted tohave channels at the cartilage cap and bone portion interface, such asosteochondral plugs (12, 24, or 32) illustrated in FIG. 2-FIG. 4. Beforebeing inserted into a holder, the length of the bone portion of theosteochondral plug was measured. Then, the osteochondral plug, e.g. astep cylindrical plug with 10 mm diameter at the bone portion, wasinserted into a holder (61), with the cartilage cap positioned to faceup and the bottom of the bone portion was supported by the custom madebolt (60) as illustrated in FIG. 8(e). The length of the osteochondralplug protruding above the top of the holder was adjusted by the custommade bolt. Then four set screws (57), preferably oriented 90 degreesapart, were engaged to further secure the osteochondral plug within theholder (61) and to adjust the superficial surface of the cartilage capon the osteochondral plug to be parallel to the bottom surface of thecustom made bolt (60). The holder was fixed horizontally, i.e. withcenterline of the osteochondral plug being parallel to the horizontaldirection, into the clamp that was fixed on the x-y table of thedrilling/milling machine so that it can move in a horizontal or crossdirection. One set of slots (62) on the holder (61) was positioneddirectly facing the drill bit. The diameter of the drill bit was chosento be 5 mm. The center of the drilling on the graft was set to be 3 mmlower than the cartilage/bone interface along the longitudinal directionof the osteochondral plug. The drill bit passed the top slot and drilledthrough the bone portion to form a through channel. Then the channel wasfurther milled to obtain a flat surface within the channel at thecartilage/bone interface to expose the deep region of the cartilage cap.

After finishing crafting the first channel, the holder withosteochondral plug inside was rotated 90 degrees to expose the secondset of slots (62). Then the second channel was crafted using the sameprocedure that was used to cut the first channel. During crafting,isotonic saline was sprayed on the graft through a cooling systeminstalled on the milling/drilling machine.

Example 4

Osteochondral Plug with Multiple Channels or a Slot at theCartilage/Bone Interface

The osteochondral plugs, crafted to be straight, step cylindrical, ordumbbell shape as illustrated in Example 1 can be further crafted tohave multiple channels or a slot at the cartilage cap and bone portioninterface, such as osteochondral plugs (16 or 18; 26 or 27; 34 or 35)illustrated in FIG. 2-FIG. 4. Before being inserted into a holder, thelength of the bone portion of the osteochondral plug was measured. Then,the osteochondral plug, e.g. a step cylindrical plug with 10 mm diameterat bone portion, was inserted into a holder (54), with the cartilage cappositioned to face up and the bottom of the bone portion was supportedby the custom made bolt (60) as illustrated in FIG. 9(e). The length ofthe osteochondral plug which protruded above the top of the holder wasadjusted by the custom made bolt. Then four set screws (57), preferablyoriented 90 degrees apart, were engaged to further secure theosteochondral plug within the holder (54) and to adjust the superficialsurface of the cartilage cap on the osteochondral plug to be parallel tothe bottom surface of the custom made bolt (60). The holder was fixedhorizontally, i.e. with centerline of the osteochondral plug beingparallel to the horizontal direction, into the clamp that was fixed onthe x-y table of the drilling/milling machine so that it can move in ahorizontal or cross direction. The set of slots (56) was positioneddirectly facing the drill/mill bit. The diameter of the drill bit waschosen to be 2 mm. For osteochondral plugs with multiple channels (16,26, or 34) as illustrated in FIG. 2-FIG. 4, the center of the firstdrilling on the graft was set to be the cross of the center along thelength of the slot (56) on the holder and 1 mm lower than thecartilage/bone interface along the longitudinal direction of theosteochondral plug. The drill bit passed through the slot (56) anddrilled through the bone portion at the cartilage/bone interface.

Then the rest of the channels were created along the length of the slot.The distance between the centers of the channels was kept at 2.5 mm. Forosteochondral plugs with a slot (18, 27, or 35) as illustrated in FIG.2-FIG. 4, the center of the first drilling on the graft was set to bethe cross of the center along the length of the slot on the holder and 1mm lower than the cartilage/bone interface along the longitudinaldirection of the osteochondral plug. The drill bit passed though theslot (56) and drilled through the bone portion at the cartilage/boneinterface. Then the drill bit was replaced with a same diameter millbit. The slot (19) on the osteochondral plug (18, 27, or 35) was createdby milling along the length of the slot (56) on the holder. The totallength of the slot on the osteochondral plug was 6 mm.

Example 5 Embossing of Circumferential Surface of the Cartilage Cap

The circumferential area of the cartilage portion of an osteochondralplug (as illustrated in example 1-4) or a cartilage disc can be furthercrafted to maximize the circumferential surface and contact areasbetween the recipient articular cartilage being repaired and thearticular cartilage graft, as illustrated in FIG. 6, to facilitateintegration of the graft tissue to the recipient tissue. A craftedosteochondral plug, such as plug (38), with tapered cylindricalcartilage cap, was further crafted to maximize circumferential surfacearea by embossing. A custom made tapered cylindrical stainless steeldie, which had a cross line pattern along the longitudinal and thecircumferential direction and with 1 mm distance between the lines, wasmounted on the cutting tool fixture of the lathe. The osteochondral plugwas fixed in a holder that held the bone portion of the plug inside. Theentire cartilage cap protruded outside of the holder. The holder wasfixed on the headstock of the lathe. The headstock was set to turn atlow speed and the die was push against the cartilage cap until a 360degree rotation was obtained.

Example 6 Microperforation of Circumferential Surface of the CartilageCap

The circumferential area of the cartilage portion of an osteochondralplug (as illustrated in Example 1-Example 4) or a cartilage disc can bemicroperforated to facilitate in situ cell migration from thesurrounding tissue to the cartilage graft. The osteochondral plug wasfixed in a holder that held the bone portion of the plug inside. Theentire cartilage cap protruded outside of the holder. The holder wasfixed horizontally, i.e. with centerline of the osteochondral plug beingparallel to the horizontal direction, into the clamp fixed on the x-ytable of the drilling/milling machine so that it could move horizontal6r cross direction. A comb of custom made needles, with outer diameterof 350 μm and 1 mm apart, was fixed on the chuck of the drilling/millingmachine with a custom made adaptor. The total width of the comb was 9mm. The punch line was set to be the half of the depth of the cartilagecap and parallel to the cartilage/bone interface. The comb of needlespassed through the entire cartilage cap.

Example 7 Cleaning and Disinfecting an Osteochondral Plug UsingCentrifugal Force

The distal end of a human femur was procured from a suitable donor,transported on wet ice to the processing facility. A picture was takenand was superimposed on a customer made grid/coordinate system to createa map of the human femoral condyle. The femoral condyle end was “cored”with a coring device or drilled with a hollow cylindrical drill bit toproduce multiple cylindrical osteochondral plugs with diameter rangefrom 5-20 mm and the length of the bone portion from 5-20 mm. Thecoordinate of each individual cylindrical plug was recorded according tothe map. The osteochondral plugs were further crafted into stepcylindrical shape and with a slot at the cartilage and bone interface asillustrated in Example 4. The crafted osteochondral plugs with diametersof 14 mm at the cartilage portion and diameter of 10 mm at the boneportion were placed in a processing chamber (75 in FIG. 13a ). Theinferior surface facing the osteochondral bone portion of the cartilagecap was placed against the top surface the porous ring (85) asillustrated in FIG. 13 a.

The bone portion of each osteochondral plug was inserted through thecenter hole of the porous ceramic ring (85) and fit into the bottomportion of the step cylinder hole with the rubber ring (89) on theperipheral surface that created a tight seal. After closing two caps (76and 79) at the top and bottom of the processing chamber, the chamber wascentrifuged at 1000 rcf for 15 minutes at ambient temperature. The bonemarrow contained in the cancellous bone part of the osteochondral plugwas induced to migrate into the bottom of the processing chamber anddiscarded. Two hundred and fifty milliliters of AlloWash® solution wastransferred into the top portion of the processing chamber. The chamberwas centrifuged at 1000 rcf for 1 hour to force the fluid pass throughthe grafts. Then the solution in both the top and the bottom portion ofthe chamber was removed and the bottom cap was closed. Two hundred andfifty milliliters of sterile distilled water containing antibiotics weretransferred into the top portion of the chamber. The chamber wascentrifuged at 1000 rcf for 30 minutes. The solution in both the top andthe bottom portion of the chamber was removed and the bottom cap wasclosed. Two hundred and fifty milliliters of isotonic saline solutionwas transferred into the top portion of the processing chamber. Thechamber was centrifuged at 1000 rcf for 15 minutes. After twice salinewash, the osteochondral plugs were ready for devitalization process.

Example 8 Cleaning and Disinfecting a Hemicondyle Using Vacuum Pressureand Sonication

The distal end of a human femur was procured from a suitable donor,transported on wet ice to the processing facility, and the condyle endwas bisected into two hemicondyles. Each hemicondyle was placed in aglass container containing 1 liter of AlloWash solution and sonicated at100 Hz for 2 hours. After sonication, the hemicondyle and AlloWash®solution was transferred into a processing chamber similar to the oneshown in FIG. 14. The bone portion of each hemicondyle was inserted intoan insert that had a large center hole with the rubber ring (created atight seal). The entire hemicondyle was immersed in the processingsolution. The bottom port of the chamber (78) was connected to tubingthat led to a collection beaker (94), which was connected to a pump(95). The pump applied a vacuum pressure between about 0 to about 20 MPato the space inside of the chamber. After vacuuming for 2 hours, allAlloWash® solution was pulled out of the chamber. One liter of sterileultra-pure water containing antibiotics was transferred into the topportion of the chamber. Vacuum pressure between about 0 to about 20 MPawas applied to the chamber for 1 hour. The solution was pulled into thebottom portion of the chamber and removed by vacuuming. One liter ofsterile ultra-pure water was transferred into the top portion of theprocessing chamber. Vacuum pressure between about 0 to about 20 MPa wasapplied to the chamber for 30 minutes. The solution was pulled into thebottom portion of the chamber and removed by vacuuming. After washingfor two more times, the hemicondyle was ready for devitalizationprocess.

Example 9 Devitalizing an Osteochondral Plug Using Centrifugal Force

Ten cleaned and disinfected step cylindrical osteochondral plugs withchannels as illustrated in Example 2, with diameter of 14 mm at thecartilage portion and diameter of 10 mm at the bone portion, werepositioned in a processing chamber (75 in FIG. 13a ). The bone portionof each osteochondral plug was inserted through the center hole of theporous ceramic ring (85) and fit into the bottom portion of the stepcylinder hole with the rubber ring (89) on the peripheral surface tocreate a tight seal. One hundred milliliters of pretreatment solutioncontaining 1 unit/mL of chondroitinase ABC in Tris/NaAc buffer wastransferred into the top part of the chamber. The chamber wascentrifuged at 1000 rcf for 6 hours at 37° C. The pretreatment solutionin both the top and the bottom portion of the chamber was removed andthe bottom cap (79) was closed. One hundred milliliters of isotonicsaline solution was transferred into the top portion of the processingchamber. The chamber was centrifuged at 1000 rcf for 15 minutes. Aftertwo more saline washes, five hundred milliliters of extracting solutionwas transferred into the top portion of the processing chamber (FIG. 13a). The extracting solution consisted of 50 mM Tris-HCl/Tris base (pH8.0), 2 mM MgCl₂, 16 mM N-lauroyl sarcosinate, 12 units/mL ofendonuclease (Benzonase®, EM Industries, Inc.), and antibioticssufficient to disinfect the tissue. The amount of endonuclease includedin the solution was calculated based on the weight of tissue to bedevitalized and the total volume of the extracting solution. Theprocessing chamber containing osteochondral plugs was centrifuged at 37°C. for 12 hours utilizing 1000 rcf to facilitate penetration of thefluid into the osteochondral plugs. Following completion of thedevitalization process, the chamber was drained of extracting solutionand replaced with 500 mL of isotonic saline. The chamber was centrifugedat 1000 rcf for 30 minutes. The saline wash was repeated two more times.

Next, the chamber was drained of saline and 250 mL of 77% (v/v) glycerolwas transferred into the top portion of the chamber. The chamber wasincubated and centrifuged at 1000 rcf for 2 hours at ambienttemperature. The glycerol was drained from the chamber. The devitalizedosteochondral plugs were transferred into an inner bag (145 in FIG. 18b(e)) with two ports (147) that sealed with Luer lock caps (148), sealedunder vacuum on one edge (146), placed in an outer bag (150) and sealed.Then, the osteochondral plugs in storage bags were sent for gammairradiation at about 15 to about 18 kGy or stored at −80° C. Samplesfrom devitalized osteochondral plugs were used for histology assessment,DNA quantification, or sulfated glycosaminoglycan (GAG) quantification.

Example 10 Devitalizing a Fibrocartilage Disc Using a Fluid Flow ThroughSystem

Ten fibrocartilage discs isolated from menisci of a cadaver donor, 10 mmin diameter, were positioned into the slots on the stainless steelporous platens on an insert (274) in a processing chamber as illustratedin FIG. 16a . The superficial surfaces of all discs were parallel to thefluid flow direction. The processing chamber was connected to themedical grade disposable tubing with 3-way stopcocks inserted in-line, aperistaltic pump and, processing solution reservoirs. The Luer lock (92)and the lid (97) were screwed down tightly to engage the o-ring therebyeliminating leakage from the chamber (96). The hydrophobic adsorbentresin and anion exchange resin were added to the resin chamber (102).There was an o-ring at the top and bottom of the resin chamber to ensurea secure fit between the resin chamber and the resin housing to forcethe flow of sterile ultra-pure water through the resin chamber. Thetubing was attached to the sipper devices (106 and 109) such that thereturn flow entered the side with the shortest spout and the outboundflow was pulled through the longest spout. The tubing was placed on therollers of the peristaltic pump and clamp lowered to hold the tubing inplace.

Then, five hundred milliliters of pretreatment solution containing 1unit/mL of chondroitinase ABC in Tris/NaAc buffer in solution reservoir(103) was drawn up from the long spout of the sipper (106), proceededthrough the port (105), continued past stopcock (113) and tubing throughthe roller assembly of the pump (95) through port (98), proceededthrough the cartilage graft and insert, then out the bottom of thechamber and through port (78) and continues past stopcocks (114 and 115and 116), then into the sipper (106) through the short spout and port(107) by using a second pump (117). This cycle continued at 250mls/minute for 16 hours at 37° C. Then one pump (95) was stopped andanother pump (117) was on until the processing chamber was empty.Stopcocks (113, 114, 115, and 116) were turned to redirect the flow toand from the sterile ultra-pure water reservoir (104) and to direct theflow through the resin housing chamber (102). The pumps (95 and 117)were turned on again and the chamber was filled by water exiting sipper(108) out the long spout, into the tubing through stopcock (113), andthrough the roller pump (95), into the processing chamber (96) throughport (98) and proceeds through the cartilage graft and insert, then outthe bottom of the chamber and through port (78) and continued paststopcock (114) which directs the flow of water into the resin chamber(102) and out of port (111) and stopcocks (115 and 116) through thetubing and into sipper (109) via the short spout and port (110) and intothe water reservoir (104) by using a second pump (117). This cyclecontinued at 250 mls/minute for 16 hours at ambient temperature. Thepressure within the processing chamber was monitored by a pressure gauge(100) that was connected to a port (99). The pretreatment solutionreservoir was replaced by an extracting solution reservoir. Afterremoving water from processing chamber, the stopcocks connected to thereservoir containing 500 milliliters of extracting solution was openedand the extracting solution proceeded through the processing chamber at250 mls/minute for 16 hours at ambient temperature.

The extracting solution consisted of 50 mM Tris-HCl/Tris Base (pH 8.0),2 mM MgCl₂, 0.5% CHAPS, 12 units/mL of endonuclease (Benzonase®, EMIndustries, Inc.), and antibiotics sufficient to disinfect the tissue.

Following completion of the devitalization process, the chamber wasdrained of extracting solution and the stopcock connected to thereservoir containing sterile ultra-pure water was opened. Ultra-purewater proceeded through the processing chamber at 250 mls/minute for 16hours at ambient temperature. The processing chamber was drained ofwater and the water reservoir was replaced by a storage solutionreservoir. The stopcock connected to the reservoir containing 500 mL of77% (v/v) glycerol was opened. Glycerol proceeded through the processingchamber at 50 mls/minute for 6 hours at ambient temperature. Thenglycerol was drained from the chamber. The devitalized fibrocartilagediscs were transferred into an inner bag (145 in FIG. 18b (e)) with twoports (147) that sealed with Luer lock caps (148), sealed under vacuumon one edge (146), placed in an outer bag (150) and sealed. Then, thefibrocartilage discs in storage bags were sent for gamma irradiation atabout 15 to about 18 kGy and stored at −80° C. Samples from devitalizedfibrocartilage discs were used for histology assessment, DNAquantification, or sulfated glycosaminoglycan (GAG) quantification.

Example 11 Devitalizing Articular Cartilage Slices in an Orbital Shaker

Twenty articular cartilage slices isolated from femoral condyle and cutto be 350-500 micrometer in thickness and 5 to 10 mm in diameter wereindividually placed in 20 microcentrifuge tubes separately. Onemilliliter of isotonic saline solution was transferred into each tube.The microcentrifuge tubes were incubated at 37° C. in an orbital shakerfor 15 minutes at 1000 rpm. After two more saline washes, one milliliterof extracting solution was transferred into each microcentrifuge tube.The extracting solution consisted of 50 mM Tris-HCl/Tris Base (pH 8.0),2 mM MgCl₂, 16 mM N-lauroyl sarcosinate, 12 units/mL of endonuclease(Benzonase®, EM Industries, Inc.), and antibiotics sufficient todisinfect the tissue. The microcentrifuge tubes containing articularcartilage slices were incubated at 37° C. in an orbital shaker for 16hours at 1000 rpm.

Following completion of the devitalization process, the tubes weredrained of the extracting solution and replaced with 1 mL of isotonicsaline. The tubes were incubated at 37° C. in an orbital shaker for 15minutes at 1000 rpm. The saline wash was repeated two more times. Thetubes were drained of extracting solution and replaced with 1 mL of 77%(v/v) glycerol. The tubes were incubated at 37° C. in an orbital shakerfor 2 hours at 1000 rpm at ambient temperature.

The devitalized articular cartilage slices were then transferred into aninner bag (145 in 18 b(e)) with two ports (147) that sealed with Luerlock caps (148), sealed under vacuum on one edge (146), placed in anouter bag (150) and sealed. Samples from devitalized fibrocartilagediscs were used for histology assessment, DNA quantification, orsulfated glycosaminoglycan (GAG) quantification.

Example 12 Devitalizing Osteochondral Plugs and Cartilage Slices UsingCyclic Hydrodynamic Pressure

Five cleaned and disinfected osteochondral plugs, with diameter of 14 mmat the cartilage portion and diameter of 10 mm at the bone portion, andten articular cartilage slices, with diameter of 14 mm and thickness of500 micrometer each, were positioned in a processing chamber (FIG. 17a). The cartilage slices were stacked together between two ceramic porousplatens that had the curvature of target defect site, and placed withina confining ring (124). The bone portion of each osteochondral plug wasinserted through the center hole of the porous stainless steel platenand fit into the bottom portion of the step cylinder hole with therubber ring on the peripheral surface to create a tight seal. Fivehundred milliliters of pretreatment solution containing 1 unit/mL ofchondroitinase ABC in Tris/NaAc buffer was transferred into theprocessing chamber, as well as the rigid tubing and the bottom part ofthe pressurization chamber (FIG. 17a ). Compressed air/gas was driven bya piston (132) and passed through the connector (286) to compress theflexible membrane (193). The piston was driven by a computer controlledcam to move up and down to create a cyclic pressure on the flexiblemembrane that transferred the pressure to the processing chamber. Thepressure was monitored using two pressure gauges (100) and regulated bytwo valves (131). The compressed air/gas was made of sterile 5% CO₂ inair.

The osteochondral plugs and cartilage discs were pre-treated withchondroitinase ABC under cycles of hydrodynamic pressure of 0 and 6 MPafor 6 hours at frequency of 1 Hz and at 37° C. The pretreatment solutionin the processing chamber was removed. Five hundred milliliters ofisotonic saline solution was transferred into the processing chamber.The grafts were then pressurized again under cyclic hydrodynamicpressure for 1 hour. After the saline drained from the processingchamber, five hundred milliliters of extracting solution was transferredinto the processing chamber. The extracting solution consisted of 50 mMTris-HCl/Tris Base (pH 8.0), 2 mM MgCl₂, 0.5% CHAPS, 12 units/mL ofendonuclease (Benzonase®, EM Industries, Inc.), and antibioticssufficient to disinfect the tissue. The osteochondral plugs andcartilage slices were processed under cycles of hydrodynamic pressure of0 and 6 MPa for 16 hours at ambient temperature.

Following completion of the devitalization process, the processingchamber was drained of extracting solution and replaced with 500 mL ofisotonic saline. The grafts were pressurized again under cyclichydrodynamic pressure for 1 hour. The saline wash was repeated two moretimes. The chamber was drained of saline and 500 mL of 77% (v/v)glycerol was transferred into the processing chamber. The grafts werepressurized again under cyclic hydrodynamic pressure for 2 hours atambient temperature. Then, the glycerol was drained from the chamber.

The devitalized osteochondral plugs or the stack of cartilage slicesalong with the contoured porous platen were transferred into an innersealed box (141) and the inner box was placed in an outer box (143) andsealed (c in FIG. 18b ). The osteochondral plugs in storage boxes weresent for gamma irradiation at about 15 to about 18 kGy and/or stored at−80° C. Samples from devitalized osteochondral plugs or cartilage sliceswere used for histology assessment, DNA quantification, or sulfatedglycosaminoglycan (GAG) quantification.

Example 13

Devitalization of Articular Cartilage Using Chondroitinase ABC and/orCHAPS made of Benzonase on a Shaker

Frozen human articular cartilage obtained from cadaver with donorconsent was used for the experiments. The 5-7 mm diameter cartilagediscs without subchondral bone were pretreated with a pretreatmentsolution composed of 1 unit/mL of chondriotinaseABC in 50 mM Tris/60 mMNaAc buffer supplemented with protease inhibitors and bovine serumalbumin at 37° C. and 1,000 rpm on a shaker for 24 hours. The cartilagediscs were washed with isotonic saline for 15 minutes at 37° C. for atotal of three times. Two samples were stored at 4° C. as chondroitinasecontrols. The rest of samples were devitalized in an extractingsolution, composed of 0.5% CHAPS, 11.5 units/mL Benzonase, 50 mM Tris,and 2 mM MgCl₂, at 37° C. and 1000 rpm in a shaker for 24 hours. Thecartilage samples were washed twice with isotonic saline for 1 hour at37° C.

The resulting cartilage was used for DNA, GAG quantification,Haematoxylin & Eosin and Safranin O staining. A Quant-it PicoGreen dsDNAkit was used to quantify the residual DNA in the cartilage. The GAGcontent was quantified by dimethylmethylene blue (DMMB) assay. FIG. 38illustrates the amount of dsDNA in cartilage detected with PicoGreenreagents. The percentage of DNA reduction was relative to thecryopreserved cartilage grafts from the same donor.

The groups treated with chondroitinase or CHAPS/Benzonase showedsignificantly lower residual dsDNA compared to cryopreserved control.The combination of chondroitinase ABC and CHAPS/Benzonase gave the mostDNA reduction (>98%).

The histology sections, stained with Haematoxylin & Eosin and SafraninO, showed that significant reduction of nucleus staining was found incartilage groups treated with chondroitinase ABC and CHAPS/Benzonase.Inter-territorial matrix removal was found in cartilage treated withchondroitinase ABC and CHAPS/Benzonase, while territorial matrixreduction was found at the surfaces that were exposed to thepretreatment or extracting solution directly (FIG. 39).

Example 14

Microperforation of the Cartilage Cap with Agarose Bead Immobilized TPCKTrypsin after Devitalization

After devitalization, the cartilage portion of an osteochondral plug (asillustrated in Example 1-Example 4) or a cartilage disc can bemicroperforated to facilitate recellularization in vitro, vivo, and insitu. Five cylindrical osteochondral plugs, 7 mm in diameter and 10 mmin length, were placed in a sterile glass beaker. Five milliliters ofagarose beads immobilized with TPCK trypsin (Pierce, Rockford, Ill.)were washed with a 0.1 NH₄HCO₃ (pH 8.0) digesting buffer. The beads werethen resuspended in 14 ml of the digest buffer, mixed, and transferredinto a beaker with osteochondral plugs. The beaker was then placed on anorbital shaker at 37° C. for 60 minutes.

During the incubation period, the beaker was taken out of the incubatorevery 15 minutes, sonicated for 2 minutes at 37° C., and returned backto the orbital shaker in the incubator. After 60 minutes of incubationand agitation, the osteochondral plugs were removed from the trypsinbead solution and placed individually in a clean 15 ml conical tube withcartilage cap facing down. The osteochondral plugs were spun at 400 rcffor 10 minutes to remove the excessive fluid.

Then the osteochondral plugs were transferred into a clean sterilebeaker and incubated with 30 ml of DMEM supplemented with 10% heatinactivated FBS or human serum for 15 minutes to inactivate the trypsinactivity. This trypsin inactivation step was repeated twice with freshDMEM supplemented with 10% heat inactivated FBS or human serum. Next,the osteochondral plugs were washed with phosphate buffered saline threetimes, and placed individually in a clean 15 ml conical tube with thecartilage cap facing down. The osteochondral plugs were spun at 400 rcffor 10 minute to remove excessive fluid.

Example 15 Bioactive Growth Supplements Coating on an Osteochondral Plug

Carboxylic acid groups of Heparin (sodium alt, 170 USP units/mg, SigmaAldrich) were activated with EDC (Sigma Aldrich) and NHS (SigmaAldrich). Ten milligrams of heparin was activated with 10 mg EDC/6 mgNHS in 5 ml of 0.05 M 2-morpholinoethnesulfonic acid (MES) buffer (pH5.6) for 10 minutes at 37° C. A straight cylindrical osteochondral plug(7 mm in diameter and 10 mm in length) was immersed in the activatedheparin solution and shaken at 200 rpm on an orbital shaker at ambienttemperature. After 4 hours of reaction, the osteochondral plug wasrinsed in 0.05 MES buffer and pH 7.4 phosphate-buffered saline (PBS)three times.

In order to induce chondrogenesis, the bone portion of the heparinimmobilized osteochondral plug (5) was fastened onto a plate (288) (FIG.40). The osteochondral plug was inverted and inserted into a container(289) that contained TGF-β solution. The level of TGF-β solution wasadjusted to just cover the entire cartilage cap. The cartilage cap wasincubated in the TGF-β solution and agitated at 60 rpm on an orbitalshaker for 4 hours at room temperature.

The TGF-β coated osteochondral plug was removed from the plate andtransferred into a container (290) that contained PDGF-bb in PBSsolution (0.2 mg/ml) (FIG. 41). The whole osteochondral plug wasincubated with PDGF-bb solution and agitated at 200 rpm on an orbitalshaker for 4 hours at room temperature. The bioactive growth supplementcoated osteochondral plug was transferred into a clean 15 ml centrifugetube and spun quickly to remove excessive fluid. Then the osteochondralplug was freeze dried, placed in a bottle, sealed, placed in an outercontainer, sealed again, and stored at −80° C.

Example 16

Recellularization of Osteochondral Plug In Situ with Bone Marrow

A devitalized osteochondral plug with a slot, such as the plug (35) inFIG. 4, stored in a vacuum sealed bag was retrieved and rinsed withisotonic saline. Freeze dried demineralized bone matrix was prepared.Two or three milliliter of bone marrow aspirate was obtained from thetibia and femur of one or two mice and mixed with 6 ml of heparin in TC199 and constantly mixed. The bone marrow was then filtered through adouble thickness of sterile gauze and through a 100 μm nylon filter. Thedevitalized osteochondral plug was mixed with 10 ml of the filtered bonemarrow until implantation to facilitate the bone marrow stromal cellattachment. The osteochondral plug, secured at the bottom of a 15 mlconical tube and mixed with the bone marrow suspension, was spun under acentrifugal force to promote further cell attachment. The demineralizedbone matrix was then mixed with the filtered bone marrow at 1:1 ratio(volume:volume). Next, the demineralized bone matrix and bone marrowmixture was inserted into the slot on the osteochondral plug. Then theosteochondral plug was ready for implantation. Optionally, right beforeimplantation, the demineralized bone matrix and bone marrow mixture canbe also inserted into the bore created at the recipient defect site. Theamount of cell attachment and cell viability were analyzed.

Example 17

Recellularization Hyaline Cartilage Disc In Situ with Chondrocyte

Autologous or allogeneic chondrocytes were isolated from non-loadbearing femoral condyle and propagated in vitro in culture media thatwas composed of Dulbecco's Modified Essential Medium (DMEM) supplementedwith 10% FBS, non-essential amino acid, 40 μg/ml proline, andantibiotics (100 U/ml penicillin and 100 μg/ml streptomycin, Invitrogen)for between 3-5 passages. A devitalized cartilage disc stored in avacuum sealed bag was retrieved and rinsed with isotonic saline. Thecartilage disc has a bore in the center, and the depth of which reachesthe middle region along the depth. Cultured chondrocytes weretrypsinized from the culture flask and suspended in culture mediasupplemented with 50 μg/ml ascorbate at 10×10⁶cell/ml density. Thedevitalized human hyaline cartilage disc was mixed with the 1.5 ml ofthe cell suspension in a 2 ml tube on a rotator located in an incubatoror water bath. The cartilage and the autologous chondrocyte suspensionwere spun to promote further cell attachment. In addition, thedemineralized bone matrix was then mixed with the chondrocyte at 1:1ratio (volume:volume). Next, the demineralized bone matrix andchondrocyte mixture was inserted into the bore in the cartilage disc.The in situ recellularized cartilage disc was ready to be implanted. Theamount of cell attachment and cell viability were then analyzed.

Example 18

Recellularization Fibrocartilage Cartilage Slices In Situ withAllogeneic Stromal Cells from Adipose Tissue

Adipose tissue was obtained from a donor. The adipose tissue was rinsedwith Hanks' balanced salt solution containing antibiotics (100 U/mlpenicillin and 100 U/ml streptomycin) and 2.5 μg/ml amphotericin B. Toisolate stromal cells, the adipose tissue was digested for 2 hours on ashaker at 37° C. in HBSS containing 0.2% collagenase (Sigma, St Louis,Mo.) and centrifuged at 1200 rcf for 10 minutes to obtain a high-densitycell pellet. The cell pellet was re-suspended in red blood cell lysisbuffer for 10 min at room temperature. The stromal cell pellet wascollected by centrifugation, as described above, and re-suspended in achondrogenic media, which was composed of DMEM (Invitrogen), 10% serum,10 ng/ml TGF-β1, 1% ITS (10 μg/ml insulin, 5.5 μg/ml transferrin, 5ng/ml selenium, 0.5 mg/ml BSA, 4.7 μg/ml linoleic acid; Sigma;), 50μg/ml ascorbate-2-phosphate, 40 μg/ml proline, 100 μg/ml pyruvate, and100 U/ml penicillin and 100 μg/ml streptomycin (all from Invitrogen) ata cell density of 2×10⁶/ml.

Devitalized human fibrocartilage slices stored in a vacuum sealed bagwas retrieved and rinsed with isotonic saline. Each individual slice ofcartilage from the same package was placed in each well of the 24-wellplate. Optionally, a cloning cylinder with grease was place on top ofthe cartilage slice to create a seal at the peripheral. Stromal cellsfrom adipose tissue were seeded on top of the cartilage slice within thecloning cylinder. The whole plate was centrifuged at 400 g for 5 min tofacilitate the cell attachment. The cartilage slices are bonded betweenadjacent slices using a bonding agent and stack together. Then, the insitu recellularized cartilage slices were ready for implantation. Theamount of cell attachment and cell viability were analyzed.

Example 19

Recellularization Hyaline Cartilage Slices In Situ with AllogeneicStromal Cells from Fibrous Synovium

Fibrous synovium was harvested from the inner side of the lateral jointcapsule, which overlays the noncartilage areas of the lateral condylesof the femur from cadaver donors. The tissue was minced to pieces with asurgical blade, washed thoroughly with phosphate buffered saline (PBS),and digested in a collagenase solution (3 mg/ml collagenase D; RocheDiagnostics, Mannheim, Germany) in α-minimum essential medium(Invitrogen, Carlsbad, Calif.) at 37° C. After 3 hours, digested cellswere filtered through a 70-μm nylon filter (Becton Dickinson, FranklinLakes, N.J.). Nucleated cells from the tissues were plated at 10³cells/cm² in a T-75 flask and cultured in DMEM, 10% FBS, 100 U/mlpenicillin and 100 μg/ml streptomycin (all from Invitrogen), and 1 ng/mLbasic fibroblast growth factor (bFGF) for 14 days before any passages.The same seeding density and media was kept for future passages. Then,passage 3 stromal cells were trypsinized and suspended in chondrogenicmedia, which was composed of DMEM (Invitrogen), 10% serum, 10 ng/mlTGF-β1, 1% ITS (10 μg/ml insulin, 5.5 μg/ml transferrin, 5 ng/mlselenium, 0.5 mg/ml BSA, 4.7 μg/ml linoleic acid; Sigma;), 50 μg/mlascorbate-2-phosphate, 40 μg/ml proline, 100 μg/ml pyruvate, and 100U/ml penicillin and 100 μg/ml streptomycin (all from Invitrogen) at acell density of 2×10⁶/ml.

Devitalized human hyaline cartilage slices stored in a vacuum sealed bagwas retrieved and rinsed with isotonic saline. Each individual slice ofcartilage from the same package was placed in each well of the 24-wellplate. One milliliter of the stromal cell suspension was added in eachwell of the 24-well plate. The plate was placed on a shaker and kept at37° C. until implantation. Optionally, the whole plate was centrifugedat 400 g for 5 min to facilitate cell attachment. Then, the in siturecellularized cartilage slices were ready for implantation. The amountof cell attachment and cell viability were analyzed.

Example 20 Recellularization In Vivo in Muscle

A devitalized rabbit osteochondral plug stored in a vacuum sealed bag isretrieved and rinsed with isotonic saline. The devitalized cartilagegraft is implanted in a muscle pouch of a nude mouse for 3 months. Thenthe cartilage disc is retrieved from the muscle, the excessive fibroustissue surrounding the recellularized cartilage graft is trimmed off,and rinsed with isotonic saline. The in vivo recellularized rabbitosteochondral plug is analyzed for cellular infiltration byimmunostaining.

Example 21 Recellularization In Vivo in a Fat Pad

A devitalized human hyaline cartilage disc without subchondral boneattached and stored in a vacuum sealed bag is retrieved and rinsed withisotonic saline. The devitalized cartilage graft is implanted in theepididymal fat pad of a nude mouse for 3 months. Then the cartilage discis retrieved from the fat pad, trimmed off the excessive fibrous tissuesurrounding the recellularized cartilage graft, and rinsed with isotonicsaline. The in vivo recellularized cartilage disc is analyzed forcellular infiltration by immunostaining.

Example 22

Recellularization of Osteochondral Plug In Vitro with Allogeneic StromalCells from Synovium

A devitalized human osteochondral plug stored in a vacuum sealed bag isretrieved and rinsed with isotonic saline. Each individual osteochondralplug is placed in a 15 ml conical tube with a custom made cap that isconnected to an air/gas filter. Allogeneic stromal cells from synovium,as illustrated in Example 19, are suspended in Dulbecco's ModifiedEssential Medium (DMEM) supplemented with 10% FBS, and antibiotics (100U/ml penicillin and 100 μg/ml streptomycin, Invitrogen) at a density of2×10⁶ cells/ml, and added into the tube to immerse the entireosteochondral plug. Then, the tube is placed on a roller, transferredinto an incubator, and cultured for 24 hours. Optionally, the cellsuspension and the osteochondral plug are centrifuged to facilitate cellattachment. After 24 hours of culture on a roller, the osteochondralplug is transferred into a bioreactor as illustrated in FIG. 28.

Then, the cartilage cap is placed within a confining ring (204) andsandwiched between a top porous platens (226) made of porous titaniumand a bottom porous ring (241) made of cancellous bone. The entireosteochondral plug is supported by the supporting ring (248) andcompressed with a loading shaft connected to a damping spring. Thecartilage cap is placed within the top well, while the bone portion isplaced in the bottom well of the bioreactor. The culture media in thetop well of the bioreactor is chondrogenic media, which is composed ofDMEM (Invitrogen), 10% serum, 10 ng/ml TGF-β1, 1% ITS (10 μg/ml insulin,5.5 μg/ml transferrin, 5 ng/ml selenium, 0.5 mg/ml BSA, 4.7 μg/mllinoleic acid; Sigma;), 50 μg/ml ascorbate-2-phosphate, 40 μg/mlproline, 100 μg/ml pyruvate, and 100 U/ml penicillin and 100 μg/mlstreptomycin. The culture media in the bottom well of the bioreactor isosteogenic, and is composed of DMEM (Invitrogen), 10% serum, 100 nMdexamethasone, 10 mM β-glycerophosphate, 50 μg/ml ascorbate-2-phosphate(Sigma), and antibiotics (100 U/ml penicillin and 100 μg/mlstreptomycin, Invitrogen). The compressive stress is cycled between 0-6MPa that is controlled by the load cell and the movement of the loadingshaft through a computer. The entire bioreactor is fit into anincubator. The media is circulated between the bioreactor and two mediareservoirs that are pumped with filtered 5% CO₂ in air. The cycliccompression is conducted for 8 hrs per day. After 4 weeks of culture,the cartilage graft is ready to be transplanted. The cell morphology,viability, extracellular matrix synthesis are analyzed.

Example 23

Recellularization of Osteochondral Plug In Vitro with Allogeneic StromalCells from Adipose Tissue, Create Contour, Load Opposing Plugs withLoading Shaft

Two devitalized human osteochondral plug with gaps, as illustrated inFIG. 4 plug (30 a) and stored in a vacuum sealed bag, are retrieved andrinsed with isotonic saline. Each individual osteochondral plug isplaced in a 15 ml conical tube with a custom made cap that is connectedto an air/gas filter. Allogeneic stromal cells from adipose tissue, asillustrated in Example 18, are suspended in Dulbecco's ModifiedEssential Medium (DMEM) supplemented with 10% FBS, and antibiotics (100U/ml penicillin and 100 μg/ml streptomycin, Invitrogen) at a density of2×10⁶ cells/ml, added into the tube to immerse the entire osteochondralplug. Then the tube is placed on a roller, transferred into anincubator, and cultured for 24 hrs. Optionally, the cell suspension andthe osteochondral plug are centrifuged to facilitate the cellattachment.

After 24 hrs of culture on a roller, two osteochondral plugs aretransferred into a bioreactor as illustrated in FIG. 30. The bottom ofthe first osteochondral plug is supported by a supporting ring (248)which is screwed into the bottom of the culture well (162) duringcompression. The second osteochondral plug is placed on top of the firstosteochondral plug and the superficial surface of the cartilage cap ofthe osteochondral plugs are placed opposing each other. In order toobtain congruent contoured surfaces between two osteochondral plugs, aporous platen (279) with the target curvature according to the contourof the recipient joint is manufactured and placed between the cartilagecaps of the two opposing osteochondral plugs. For confined compression,cartilage caps from both osteochondral plugs are placed in a confiningring (247) (FIG. 29). The culture media in the top and bottom wells ofthe bioreactor are chondrogenic media, which is composed of DMEM(Invitrogen), 10% serum, 10 ng/ml TGF-β1, 1% ITS (10 μg/ml insulin, 5.5μg/ml transferrin, 5 ng/ml selenium, 0.5 mg/ml BSA, 4.7 μg/ml linoleicacid; Sigma;), 50 μg/ml ascorbate-2-phosphate, 40 μg/ml proline, 100μg/ml pyruvate, and 100 U/ml penicillin and 100 μg/ml streptomycin. Theloading shaft is directly compressed on the bone portion of the secondosteochondral plug, a solid bead (243), and a porous platen (226) toensure the center line of the loading shaft is parallel to thecenterline of the osteochondral plugs to be compressed. The loadingshaft is driven by a computer controlled cam and a stepper motor to moveup and down to create a cyclic compression within the bioreactor. Thecompressive stress is cycled between 0-6 MPa and is controlled by theload cell and the movement of the loading shaft through a computer. Theentire bioreactor is fit into an incubator. The media is circulatedbetween the bioreactor and two media reservoirs that are pumped withfiltered 5% CO₂ in air. The cyclic compression is conducted for 8 hrsper day. After 4 weeks of culture, the cartilage graft is ready to betransplanted. The cell morphology, viability, extracellular matrixsynthesis are analyzed.

Example 24

Recellularization of Costal Cartilage Disc In Vitro with Chondrocytes,Cultured Under Fluid Pressure

Autologous chondrocytes isolated from recipient's non-load bearingfemoral condyle or from a allogeneic source were propagated in vitro inculture media that was composed of Dulbecco's Modified Essential Medium(DMEM) supplemented with 10% FBS, non-essential amino acid, 40 μg/mlproline, and antibiotics (100 U/ml penicillin and 100 μg/mlstreptomycin, Invitrogen) for between 3-5 passages. A devitalized costalcartilage disc stored in a vacuum sealed bag was retrieved and rinsedwith isotonic saline. Cultured autologous chondrocytes were trypsinizedfrom the culture flask and suspended in culture media supplemented with50 μg/ml ascorbate at a density of 10×10⁶ cell/ml. The devitalizedcartilage disc was mixed with 1.5 ml of cell suspension in a 2 ml tubeon a thermal mixer at 37° C. for about 1 hour. Then, the cartilage discwas transferred into a confining ring that had a porous platen made ofcancellous bone at the bottom and was on top of another porous platen asillustrated in FIG. 24. On top of the disc, a second porous platen madeof porous titanium was added. The cartilage disc was compressed byinducing compression on the culture media by a piston in a mediareservoir (221), which induced pressure on the cartilage graft in abioreactor filled with the culture media as illustrated in FIG. 24. Thepiston was driven by a computer controlled cam. The pressure was cycledbetween 0-2 MPa. The pressure induced compression was applied for 8 hrsper day. The entire bioreactor assembly was fit into an incubator. After14 days of culture, the top porous platen was removed. The cartilagedisc along with the bottom porous platen formed a coherent cartilagegraft and was ready to be transplanted. The cell morphology, viability,extracellular matrix synthesis were analyzed.

Example 25

Recellularization of Hyaline Cartilage Slices In Vitro with AllogeneicStromal Cells from Synovium with Fluid Pressure

Allogeneic stromal cells from synovium, as illustrated in Example 19,were suspended in chondrogenic media, which was composed of DMEM(Invitrogen), 10% serum, 10 ng/ml TGF-β1, 1% ITS (10 μg/ml insulin, 5.5μg/ml transferrin, 5 ng/ml selenium, 0.5 mg/ml BSA, 4.7 μg/ml linoleicacid; Sigma;), 50 μg/ml ascorbate-2-phosphate, 40 μg/ml proline, 100μg/ml pyruvate, and 100 U/ml penicillin and 100 μg/ml streptomycin.Devitalized human hyaline cartilage slices, stored in a vacuum sealedbag, were retrieved and rinsed with isotonic saline. Each individualslice of cartilage from the same package was placed in each well of a24-well plate. Optionally, a cloning cylinder with grease was placed ontop of the cartilage slice to create a seal at the peripheral.Allogeneic stromal cells from synovium, suspended at 2×10⁶ cells/ml,were seeded on top of the cartilage slice within the cloning cylinder.The whole plate was centrifuged at 400 g for 5 min to facilitate thecell attachment. Each individual cell-seeded slice was transferred in toa confining ring that had a porous platen made of cancellous bone at thebottom and was on top of another porous platen as illustrated in FIG.24. All the slices were stacked within the confining ring. On top of thestack, a second porous platen made of porous titanium was added. Thecartilage slices were compressed by inducing compression on the culturemedia by a piston in a media reservoir (221), which induced pressure onthe cartilage graft in a bioreactor filled with the culture media asillustrated in FIG. 24. The piston was driven by a computer controlledcam. The pressure was cycled between 0-6 MPa. The pressure inducedcompression was conducted for 8 hrs per day. The entire bioreactorassembly was fit into an incubator. After 14 days of culture, the topporous platen was removed. The cartilage slices along with the bottomporous platen formed a coherent cartilage graft and was ready to betransplanted. The cell morphology, viability, extracellular matrixsynthesis were analyzed.

Example 26

Recellularization of Hyaline Cartilage Slices In Vitro with AllogeneicStromal Cells from Bone Marrow to Create a Contour Using Air Pressure

Human allogeneic bone marrow stromal cells (BMSCs) were isolated,cultured, expanded and used for recellularization. Frozen allogeneicwhole bone marrow obtained from a commercial source was quickly thawed,washed, counted, and suspended in Dulbecco's modified Eagle medium(DMEM), 10% serum, 0.1 mM nonessential amino acids, antibiotics (100U/ml penicillin and 100 μg/ml streptomycin, Invitrogen) and 1 ng/mLbasic fibroblast growth factor (bFGF). The stromal cells were culturedin T-75 flask with cell density of 10³/ml for 3 hrs to allow adherentcells to attach. Then the non-adherent cells were washed out with DMEM.The adherent cells were cultured until near confluence. Passage 3 BMSCswere trypsinized and suspended in chondrogenic media, which was composedof DMEM (Invitrogen), 10% serum, 10 ng/ml TGF-β1, 1% ITS (10 μg/mlinsulin, 5.5 μg/ml transferrin, 5 ng/ml selenium, 0.5 mg/ml BSA, 4.7μg/ml linoleic acid; Sigma;), 50 μg/ml ascorbate-2-phosphate, 40 μg/mlproline, 100 μg/ml pyruvate, and 100 U/ml penicillin and 100 μg/mlstreptomycin.

Devitalized human hyaline cartilage slices, without subchondral boneattached and stored in a vacuum sealed bag, were retrieved and rinsedwith isotonic saline. Each individual slice of cartilage, from the samepackage, was placed in each well of the 24-well plate. Optionally, acloning cylinder with grease was place on top of the cartilage slice tocreate a seal at the peripheral. Passage 3 BMSCs, suspended at 2×10⁶cells/ml, were seeded on top of the cartilage slice within the cloningcylinder. The whole plate was centrifuged at 400 g for 5 min tofacilitate cell attachment. The BMSC seeded slices were further culturein a 24-well plate for another 24 hours. Each individual cell-seededslice was then transferred into a confining ring that had a convexporous platen made of cancellous bone at the bottom and was on top ofanother porous platen as illustrated in FIG. 23. All the slices werestacked within the confining ring. On top of the stack, a second convexporous platen made of porous titanium was added. The stacked cartilageslices were compressed by inducing compressive air towards two flexiblemembranes (172 and 193) that induce pressure on the cartilage graft abioreactor filled with the culture media (same as above) as illustratedin FIG. 23. The pressure was cycled between 0-2 MPa and induced by thefiltered 5% CO₂ in air driven by the piston and a computer controlledcam. The pressure induced compression was conducted for 8 hrs per day.The entire bioreactor assembly was fit into an incubator. After 14 daysof culture, the top convex porous platen was removed. The stack ofcartilage slices along with the bottom porous platen formed a coherentcartilage graft and was ready to be transplanted. The cell morphology,viability, extracellular matrix synthesis were analyzed.

Example 27

Recellularization of Costal Cartilage Slices and Bone Plug (to FormComposite Graft) In Vitro with Allogeneic Stromal Cells from Synovium,No Combine Culture

Cartilage slices isolated from cadaver costal cartilage, aredisinfected, cleaned, devitalized, and recellularized with allogeneicstromal cells from synovium and cultured under mechanical stimuli asillustrated in Example 25 to form a coherent stack of cartilage slices.Parallel to the cartilage slice culture, a hollow cylindrical bone plug(with same outer diameter as the cartilage discs and a center hole inthe middle), cleaned, disinfected, freeze dried and sterilized, issoaked in DMEM for 30 min. Allogeneic stromal cells from synovium,suspended at a density of 2×10⁶ cells/ml, are mixed with the bone plugon a thermal mixer overnight at 37° C. On the second day, a highlyporous calcium phosphate, obtained from a commercial source, is mixedwith the stromal cell suspension. The mixture is inserted into thecenter of the hollow cylindrical plug. The entire bone plug is furthercultured in a roller bottle or under mechanical compression similar tothe compression of osteochondral plug as illustrated in Example 22 usingosteogenic culture media. The media is composed of DMEM (Invitrogen),10% serum, 100 nM dexamethasone, 10 mM β-glycerophosphate, 50 μg/mlascorbate-2-phosphate (Sigma), and antibiotics (100 U/ml penicillin and100 μg/ml streptomycin, Invitrogen). After 4 weeks of parallel cultureof cartilage discs and the bone plug, the grafts are retrieved fromcorresponding bioreactors and are ready for transplantation. The cellmorphology, viability, extracellular matrix synthesis are analyzed.

Example 28

Recellularization of Menisci Cartilage Slices and Bone Plug (to FormComposite Graft) In Vitro with Allogeneic Stromal Cells from Synovium,Combine Cultured Under Loading with Loading Shaft

Cartilage slices isolated from cadaver menisci, are disinfected,cleaned, devitalized, and recellularized with allogeneic stromal cellsfrom synovium and cultured under mechanical stimuli as illustrated inExample 25 to form a coherent stack of cartilage slices. Parallel to thecartilage slice culture, a hollow cylindrical bone plug (with same outerdiameter as the cartilage discs and a center hole in the middle),cleaned, disinfected, freeze dried and sterilized, is soaked in DMEM for30 minutes. Allogeneic stromal cells from bone marrow, suspended at adensity of 2×10⁶ cells/ml, are mixed with the bone plug on a thermalmixer over night at 37° C. On the second day, demineralized bone matrix,from the same donor as the bone plug, is mixed with the stromal cellsuspension and the mixture is inserted into the center of the hollowcylindrical bone plug. The entire bone plug is further cultured in aroller bottle or under mechanical compression similar to the compressionof osteochondral plug as illustrated in Example 22 using osteogenicculture media. The media is composed of DMEM (Invitrogen), 10% serum,100 nM dexamethasone, 10 mM β-glycerophosphate, 50 μg/mlascorbate-2-phosphate (Sigma), and antibiotics (100 U/ml penicillin and100 μg/ml streptomycin, Invitrogen). After 1 weeks of parallel cultureof cartilage discs and the bone plug, the grafts are retrieved fromcorresponding bioreactors and transferred into another bioreactor asillustrated in FIG. 28. The bone plug is supported by the supportingring (248). The stack of cartilage slices are placed within a confiningring (204) and sandwiched between a top porous platens (226) made ofporous titanium and the bone plug. The cartilage slices are placedwithin the top well, while the bone plug is placed in the bottom well ofthe bioreactor. The culture media in the top well of the bioreactor ischondrogenic media, which is composed of DMEM (Invitrogen), 10% serum,10 ng/ml TGF-β1, 1% ITS (10 μg/ml insulin, 5.5 μg/ml transferrin, 5ng/ml selenium, 0.5 mg/ml BSA, 4.7. μg/ml linoleic acid; Sigma;), 50μg/ml ascorbate-2-phosphate, 40 μg/ml proline, 100 μg/ml pyruvate, and100 U/ml penicillin and 100 μg/ml streptomycin. The culture media in thebottom well of the bioreactor is osteogenic, which is composed of DMEM(Invitrogen), 10% serum, 100 nM dexamethasone, 10 mM β-glycerophosphate,50 μg/ml ascorbate-2-phosphate (Sigma), and antibiotics (100 U/mlpenicillin and 100 μg/ml streptomycin, Invitrogen). The compressivestress is cycled between 0-6 MPa that is controlled by the load cell andthe movement of the loading shaft through a computer. The entirebioreactor is fit into an incubator. The media is circulated between thebioreactor and two media reservoirs that are pumped with filtered 5% CO₂in air. The cyclic compression is applied for 8 hrs per day. After 3weeks of culture, a composite graft is obtained and ready to betransplanted. The cell morphology, viability, extracellular matrixsynthesis are analyzed.

Example 29 Implant Osteochondral Plug

A devitalized rabbit osteochondral plug, recellularized in vivo asillustrated in Example 20, is used for implantation. The osteochondralplug is step cylindrical and has one slot as shown in plug (35) in FIG.4. Both knee joints of a New Zealand white rabbit are exposed through amedial parapatellar longitudinal incision. The capsule is incised, andthe medial femoral condyle exposed. With the knee maximally flexed, afull-thickness bore, 3 mm in diameter and 3 mm in depth, is created inthe center of the condyle using a drill with 3 mm outside diameter. Astop is mounted on the drill bit to insure the 3 mm depth of the bore.All debris is removed from the defect with a curette and the edgecarefully cleaned with a scalpel blade. The tissue removed from thecoring is further crushed and used for later implantation. A bore iscreated on the opposing leg and remained untreated to serve as acontrol. The bore on the treated side is filled with 24 mMN-(2′-ethylaminoethyl)-4-amino-1,8-naphthalimide in gelatin solutionsupplemented with 5 μM lycopene (Sigma) for 10 minutes to stain thecartilage tissue. Meanwhile, the circumferential area of the cartilagecap of the osteochondral plug is treated with the sameN-(2′-ethylaminoethyl)-4-amino-1,8-naphthalimide solution. The crushedtissue removed from the coring is inserted into the slot on theosteochondral plug inserted. After finishing staining with thephotoactive dye, the bore in the bone portion is rinsed with isotonicsaline.

Next, part of the crushed tissue is inserted back into the bore in therecipient joint to fill the gap between the bore and the bone portion ofthe step cylinder. The osteochondral plug is transferred to the blindbore and pushed slight until interference with the surrounding cartilagetissue. A needle connected to an insertion device is inserted throughthe cartilage cap. A vacuum device is engaged to remove the air/gas andfluid trapped within the bore and forced the osteochondral plug into theblind bore. After the graft is properly inserted for 2-10 minutes, thephotoactivated dye is activated by a laser with 457 nm wave length asillustrated in FIG. 34. A 2.5 mm disc is placed at the center of thecartilage graft to protect it from the laser beam. The laser beam isdelivered through an optical fiber with a spot size of 4 mm and anintensity of ˜2 W/cm². The exposure time is about 240 seconds. Then,both knee joints are closed. The graft remains in place for 4 weeks andis analyzed.

Example 30

Implant Cartilage Disc from Menisci

A devitalized rabbit cartilage disc, isolated from menisci, is craftedto star-shaped right before implantation and recellularized in situ asin Example 17. Both knee joints of a New Zealand white rabbit areexposed through a medial parapatellar longitudinal incision. The capsuleis incised, and the medial femoral condyle exposed. With the kneemaximally flexed, a first full-thickness bore, 3 mm in diameter and 3 mmin depth, is created in the center of the condyle using a drill with 3mm outside diameter. A stop is mounted on the drill bit to insure the 3mm depth of the bore. Then a star-shaped second bore is created only atthe cartilage portion of the first bore, using a custom designed coringdevice as illustrated in FIG. 31a . All debris is removed from thedefect with a curette and the edge is carefully cleaned with a scalpelblade. A bore is created on the opposing leg and remained untreated toserve as a control. The bore on the treated side is filled with 0.1%Rose Bengal in phosphate buffered saline (PBS) and supplemented with 5μM lycopene (Sigma) for 5 minutes to stain the cartilage tissue.Meanwhile, the circumferential area of the cartilage disc is treatedwith the same Rose Bengal solution. After staining with the photoactivedye, the first bore in the bone portion is rinsed with isotonic saline.

Bone filler is made by mixing the freeze dried demineralized bone matrixwith the wet homogenized fascia at 1:1 ratio (by weight). Bone filler ispacked into the bone portion of the first bore that is created at thedefect site to provide support for the cartilage. The cartilage disc istransferred to the blind bore, fit into the star-shaped bore, and pushedslightly until interference with the surrounding cartilage tissue. Next,a needle connected to an insertion device is inserted through thecartilage disc. A vacuum device is engaged to remove the air/gas andfluid trapped within the blind bore and forces the cartilage disc intothe blind bore.

After the graft is properly inserted for 2 minutes, the photoactivateddye is activated by a laser as illustrated in FIG. 34 with 564 nm wavelength. A 2.5 mm disc is placed at the center of the cartilage graft toprotect it from the laser beam. The laser beam is delivered through anoptical fiber with a spot size of 5 mm with intensity of ˜1 W/cm². Theexposure time is about 250 seconds. Then, both knee joints are closed.The graft is remained in place for 4 weeks and is analyzed.

Example 31 Implant Hyaline Cartilage Slices

Both knee joints of a New Zealand white rabbit are exposed through amedial parapatellar longitudinal incision. The capsule is incised, andthe medial femoral condyle exposed. With the knee maximally flexed, apartial-thickness bore, 3 mm in diameter and broke the tide mark indepth, is created in the center of the condyle using a drill with 3 mmoutside diameter. A stop is mounted on the drill bit to insure the depthof the bore is slightly deeper than the cartilage tissue depth (˜1 mm).All debris is removed from the defect with a curette and the edgecarefully cleaned with a scalpel blade. A bore is created on theopposing leg and remained untreated to serve as a control. Devitalizedrabbit cartilage slices, of 250 μm thickness, are seeded with allogeneicstromal cells in situ as illustrated in Example 19, and punched to 3 mmdiameter. The bore on the treated side is filled with 0.1% riboflavin(10 mg riboflavin 5-phosphate in 10 ml 20% dextran-T-500) supplementedwith 5 μM lycopene (Sigma) for 5 minutes to stain the cartilage tissue.Meanwhile, the circumferential area of each of the cartilage slices istreated with the same riboflavin solution. After staining with thephotoactive dye, riboflavin is removed from the bore. Each individualcartilage slice is transferred, pushed into the bore against thesubchondral bone, and the slices are stacked together until reach thesame height as the surrounding tissue. The cartilage slices are bonedbetween adjacent slices using a bonding agent made of MATRIGEL® andgenipin. After the graft is properly inserted, the photoactivated dye isactivated by two ultraviolet A diodes as illustrated in FIG. 34 with 370nm wave length. A 2.5 mm disc is placed at the center of the cartilagegraft to protect it from the light beam. The light beam is deliveredthrough an optical fiber with a spot size of 4 mm and intensity of about3 mW/cm². The exposure time is about 30 minutes. Then, both knee jointsare closed. The graft are remained in place for 4 weeks and analyzed.

Example 32

Implant Cartilage Slices with Bone Plug and Calcium Phosphate CompositeCylinder

Both knee joints of a New Zealand white rabbit are exposed through amedial parapatellar longitudinal incision. The capsule is incised, andthe medial femoral condyle exposed. With the knee maximally flexed, afull-thickness bore, 3 mm in diameter and 3 mm in depth is created inthe center of the condyle using a drill with 3 mm outside diameter. Astop is mounted on the drill bit to insure the 3 mm depth of the bore.All debris is removed from the defect with a curette and the edgecarefully cleaned with a scalpel blade. A bore is created on theopposing leg and remained untreated to serve as a control. Devitalizedrabbit cartilage slices, of 250 μm thickness, are seeded with allogeneicstromal cells, stacked and cultured to form a viable coherent cartilagegraft as illustrated in Example 25, and punched to 3 mm diameter. A boneplug filled with porous tri-calcium phosphate and cultured asillustrated in Example 27 is trimmed to the length of the bone portionof the bore at the defect site. The bore on the treated side is filledwith 0.1% riboflavin (10 mg riboflavin 5-phosphate in 10 ml 20%dextran-T-500) supplemented with 5 μM lycopene (Sigma) and 5% genipinfor 5 minutes to stain the cartilage tissue. Meanwhile, thecircumferential area of each of the cartilage slices is treated with thesame riboflavin and genipin solution.

After finishing staining with the photoactive dye and crosslinkingagent, riboflavin and genipin solution is removed from the bore. Thebone plug is inserted into the bore first. Then the stack of cartilageslices is transferred to the bore, fit into the bore, and pushed slightuntil reaching the same height as the surrounding tissue. Then, thephotoactivated dye is activated by two ultraviolet A diodes asillustrated in FIG. 34 with 370 nm wave length. A 2.5 mm disc is placedat the center of the cartilage graft to protect it from the light beam.The light beam is delivered through an optical fiber with a spot size of4 mm with intensity of about 3 mW/cm². The exposure time is 30 minutes.Then, both knee joints are closed. The graft is remained in place for 4weeks and analyzed.

Example 33

Implant Cartilage Curls with a Cartilage Disc

Both knee joints of a New Zealand white rabbit are exposed through amedial parapatellar longitudinal incision. The capsule is incised, andthe medial femoral condyle exposed. With the knee maximally flexed, afull-thickness bore, 3 mm in diameter and 3 mm in depth, is created inthe center of the condyle using a drill with 3 mm outside diameter. Astop is mounted on the drill bit to insure the 3 mm depth of the bore.All debris is removed from the defect with a curette and the edgecarefully cleaned with a scalpel blade. A bore is created on theopposing leg and remained untreated to serve as a control. The bore onthe treated side is filled with 0.1% Rose Bengal in collagen solutionsupplemented with 5 μM lycopene (Sigma Aldrich) for 5 minutes to stainthe cartilage tissue. Meanwhile, the circumferential area of the rabbitcartilage disc is treated with the same Rose Bengal solution. Afterfinishing staining with the photoactive dye, the bore in the boneportion is rinsed with isotonic saline.

Next, devitalized rabbit cartilage curls are mixed with freeze driedrabbit demineralized bone matrix (v/v=1:1). Bone marrow withdraw fromthe same rabbit is used to hydrate the cartilage and DBM mixture. Thehydrated cartilage and DBM mixture is packed into the bottom portion ofthe bore to about 2 mm in depth. The cartilage disc is transferred tothe bore, fit into the bore, and pushed slightly until interference withthe surrounding cartilage tissue. A needle connected to an insertiondevice is inserted through the cartilage disc. A vacuum device isengaged to remove the air/gas and fluid trapped within the blind boreand forces the cartilage disc into the blind bore.

After the graft is properly inserted, the photoactive dye is activatedby a laser as illustrated in FIG. 34 with 564 nm wave length. A 2.5 mmdiameter non-light penetrable disc is placed at the center of thecartilage graft to protect it from the laser beam. The laser beam isdelivered through an optical fiber with a spot size of 5 mm withintensity of ˜1 W cm². The exposure time is about 250 seconds. Then,both knee joints are closed. The graft remains in place for 4 weeks andis the analyzed.

What is claimed:
 1. A shaped cartilage matrix isolated from a human oranimal, comprising cartilage which is crafted to facilitatedisinfection, cleaning, devitalization, recellularization, andintegration after implantation; and wherein said cartilage matrix isisolated from whole condyles, whole plateaus, hemicondyles,hemiplateaus, femoral heads, phalanges, talus, tibia, fibula, rib,intervertebral discs, menisci, nose, or ear, wherein said cartilagematrix is in the form of osteochondral plug; wherein said osteochondralplug comprises a cartilage cap and a subchondral bone portion contactedto said cartilage cap with a tidemark at an interface between thecartilage and subschondral bone; wherein said osteochondral plug has astraight cylindrical shape, a step cylindrical shape, or a dumbbellshape; wherein the subchondral bone portion of said osteochondral pluggaps, bores, slots, or channels extending through the tidemark at thecartilage and subchondral bone interface; and wherein said modificationfacilitates disinfection, cleaning, devitalization, recellularization invivo, in situ, or in vitro, and integration after implantation.
 2. Aprocess for repairing a cartilage defect and implanting a cartilagegraft into a human or animal, comprising a. crafting the cartilagematrix of claim 1 into individual grafts; b. disinfecting and cleaningsaid cartilage graft; c. applying a pretreatment solution to saidcartilage graft; d. removing cellular debris using an extractingsolution to produce a devitalized cartilage graft; e. implanting saidcartilage graft into said cartilage defect with or without an insertiondevice; and f. sealing the implanted cartilage graft to recipienttissue; wherein said devitalized cartilage graft is recellularized withviable cells to render said tissue vital before or after saidimplanting; wherein said recellularizing devitalized cartilage graft iscarried out in vitro, in vivo, or in situ; and wherein said devitalizedcartilage graft is optionally stored between said removing cellulardebris and recellularizing steps.
 3. A repaired cartilage defectrepaired by the process of claim
 2. 4. The cartilage matrix of claim 1,wherein said osteochondral plug is crafted to have one or multiplegap(s).
 5. The cartilage matrix of claim 1, wherein said osteochondralplug is drilled and/or milled from the bottom of the subchondral boneportion to form one or more channels.
 6. The cartilage matrix of claim1, wherein said osteochondral plug is drilled and/or milled on thecylindrical surface of the subchondral bone portion at the cartilage andsubchondral bone interface to form one or more through channels.
 7. Thecartilage matrix of claims 1, wherein said osteochondral plug is drilledand/or milled on the cylindrical surface of the subchondral bone portionat the cartilage and subchondral bone interface to form one or moreslots.
 8. The cartilage matrix of claim 1, wherein the circumferentialsurface area of said cartilage cap on said osteochondral plug ismodified to facilitate the integration of graft tissue to a recipienttissue.
 9. The cartilage matrix of claim 8, wherein said cartilage capis crafted to have a star shape or tapered cylindrical shape to maximizethe circumferential surface area of said cartilage cap on saidosteochondral plug and/or to prevent rotation of said osteochondral plugin the recipient tissue.
 10. The cartilage matrix claim 8, wherein saidcartilage caps are crafted to increase circumferential surface area byspraying or blasting microparticles or embossing with a straight ornon-straight line pattern or cross-line pattern onto the circumferentialsurface.
 11. The cartilage matrix of claim 1, wherein thecircumferential surface and/or the superficial surface of said cartilagecap on said osteochondral plug is microperforated to facilitatedisinfection, cleaning, devitalization, recellularization in vivo, insitu, or in vitro, and the integration of graft tissue to a recipienttissue.
 12. The cartilage matrix of claim 4, wherein said cartilage capis sectioned off the subchondral bone portion to become a cartilagedisc; wherein said cartilage disc has a full or partial depth; whereinsaid full or partial depth cartilage disc has one or more gaps startingfrom the deep zone of said cartilage disc; and wherein said gaps end atthe deep, middle, or superficial zone of said cartilage cap along thedepth direction and do not penetrate the superficial surface of saidcartilage disc.
 13. The cartilage matrix of claim 5, wherein saidcartilage cap is sectioned off the subchondral bone portion to become acartilage disc; wherein said cartilage disc has a full or partial depth;and wherein said full or partial depth cartilage disc has one or morechannels starting from the deep zone of said cartilage disc; and whereinsaid channels end at the deep, middle, or superficial zone of saidcartilage disc along the depth direction and do not penetrate thesuperficial surface of said cartilage disc.
 14. The cartilage matrix ofclaim 6, wherein said cartilage cap is sectioned off the subchondralbone portion to become a cartilage disc; wherein said cartilage disc hasa full or partial depth; and wherein said full or partial depthcartilage disc has one or more through channels that are also parallelto the superficial surface of said cartilage disc and start at the deepzone of said cartilage disc; and wherein said channels occupy portionsof the deep and/or middle zone of said cartilage disc along the depthdirection and do not occupy the superficial zone of said cartilage disc.15. The cartilage matrix of claim 7, wherein said cartilage cap issectioned off the subchondral bone portion to become a cartilage disc;wherein said cartilage disc has a full or partial depth; and whereinsaid full or partial depth cartilage disc has one or more slots startingat the deep zone of said cartilage disc; and wherein said slots occupyportions of the deep and/or middle zone of said cartilage disc along thedepth direction and do not occupy the superficial zone of said cartilagedisc.
 16. The cartilage matrix of claim 1, wherein said cartilage matrixis sectioned, shaved, or skived into cartilage slices and wherein saidcartilage slices are tailored to have circular, square, triangular orstar shapes.
 17. The cartilage matrix of claim 1, wherein said cartilagematrix is shaved or skived into cartilage curls or flakes in irregularshapes.
 18. The cartilage matrix of claim 17, wherein said cartilagecurls or flakes are mixed with a carrier.
 19. A shaped cartilage matrixisolated from a human or animal, comprising cartilage which is crafted,cleaned, disinfected, and devitalized.
 20. A shaped cartilage matrixisolated from a human or animal, comprising cartilage which is crafted,cleaned, disinfected, devitalized, and recellularized.
 21. The processof claim 2, wherein said cartilage matrix is isolated to repair hyalinecartilage defects, elastic cartilage defects, or fibrocartilage defects.